3th International Workshop

“Emerging Target Therapies in Cancer:

Toward effective combinations”

October 24 to 27, 2019

Melia Las Dunas Hotel, Cuba

Index

OPENING SESSION .............................................................................. 1

Thursday October, 24th .................................................................. 1

ORAL PRESENTATIONS ....................................................................... 2

Friday October, 25th ........................................................................ 2

Saturday October, 26th.................................................................... 4

Sunday October, 27th ...................................................................... 6

ORAL SESSION ABSTRACTS ................................................................ 7

Extending the small molecule similarity principle to all levels of

biology ............................................................................................ 7

Biomarker Discovery in Cancer: Building a bridge between

preclinical and clinical research ...................................................... 8

Heberferon, combination of ifns for cancer treatment. The omics

opportunities. ................................................................................. 9

Omics approaches in glioblastoma multiforme under interferon

co-formulation treatment ............................................................ 10

OMICS-credentialing of patient-derived glioblastoma models for

pre-clinical studies ........................................................................ 11

Novel gene targets in head-and-neck cancer revealed by co-

expression networks using multi-omic data integration .............. 12

Personalized Cancer Therapy Prioritization Based on Driver

Alteration Co-occurrence Patterns ............................................... 13

Investigation of epithelial-to-mesenchymal transition mechanisms

implicated in ovarian cancer dissemination ................................. 14

Molecular taxonomy of metastatic renal cell carcinoma and its

correlation with outcome to immunotherapy ............................. 15

Steroidogenic Factor-1 is a Goldilocks transcription factor

regulating key genes implicated in tumorigenesis ....................... 16

Phosphoproteomic study of the antitumorals CIGB300 and CX-

4945. ............................................................................................. 17

Application of tensor decomposition based unsupervised feature

extraction to multi-omics data set ............................................... 18

Targeting therapeutic resistance in colorectal cancer ................. 19

Role of ABCB5 in therapeutic resistance to targeted melanoma

therapies ....................................................................................... 20

Microenvironmental influences in melanoma: a SPARC-ling affair

...................................................................................................... 21

Contribution of extracellular matrix signaling to melanoma drug

resistance ...................................................................................... 22

CD200-Expresson on Vascular Endothelial cells is mediated

through VEGF ................................................................................ 23

CIGB-247, a VEGF based active immunotherapy: Going for tumor

microenvironment ........................................................................ 24

CIGB-300: A Clinical-stage Anti-CK2 Peptide-based Drug for Cancer

Therapy ......................................................................................... 25

CE-MS in clinical proteomics: application in oncology, kidney and

cardiovascular diseases ................................................................ 26

Heberprovac, a GnRH based vaccine directed to prostate Cancer.

Clinical trials results and new challenges. .................................... 27

Overpowering multiple inhibitory immune checkpoints with a

single peptide inhibitor. ................................................................ 28

CRISPR/Cas9-mediated gene knockout of COMMD1 decreases

sensitivity of H460 and HCT-116 cell lines to the antitumoral

peptide CIGB-552. ......................................................................... 29

POSTER SESSION .............................................................................. 31

POSTER SESSION ABSTRACTS .......................................................... 35

A co-formulation of interferons type I and II enhances

temozolamide response in glioblastoma by decreasing MGMT

levels ............................................................................................. 35

Global analysis of genes expression in human glioblastoma

derived clones treated with the interferon co-formulation

HeberFERON. ................................................................................ 36

First exploration of proteomic profile regulated by the co-

formulation of type I and II interferons HeberFERON .................. 37

Clinical development of CIGB-247 active cancer immunotherapy:

Entering Phase II/III clinical trials .................................................. 38

Where VEGF should be measured? An experience obtained from

phase I clinical trials cancer patients treated with a VEGF vaccine.

...................................................................................................... 39

Antitumoral effects of a VEGF based active immunotherapy

formulated in Alum Phosphate in orthotopic ovarian tumors in

C57Bl/6 mice. ................................................................................ 40

Transcriptional evaluation of genes related to the potential

mechanism of action of the VEGF based active immunotherapy

CIGB247, in the context of CT26 ectopic tumors: Preliminary

results. .......................................................................................... 41

ELISA method for quantification of antibodies against VEGF

protein. ......................................................................................... 42

Clinical Research of CIGB-300 in patients with Locally Advanced

Cervical Cancer ............................................................................. 43

Effect of the clinical-grade Casein Kinase 2 inhibitor CIGB-300 on

NF-kB signaling in Acute Myeloid Leukemia cells ......................... 44

NPM1c+ as a putative novel target of interaction for the

antitumoral peptide CIGB-300 ...................................................... 45

MODULATION OF PD-L1 EXPRESSION BY CIGB-300 IN HUMAN

CERVICAL CANCER CELL LINES ...................................................... 46

MultiOMICs data analysis enables identification of biological

processes and pathways modulated by CIGB-300 in leukemia cells

...................................................................................................... 47

Phosphoproteomic profile modulated by the anticancer peptide

CIGB-300 in human promyelocytic leukemia (HL-60) cells. .......... 48

The influence of different peptide to increase the immunogenicity

of the GnRH vaccine for prostate cancer treatment. ................... 49

Bioequivalence of natural and synthetic VSSP using the peptide

GnRHm1/TT for the treatment of prostate cancer. ..................... 50

Establishment of an ELISA for the quantification of the CIGB-552

peptide in plasma samples from patients of ARGOS Phase I Clinical

Trial. .............................................................................................. 51

Studies of different doses of the CIGB550-E7 vaccine candidate

with a fixed dose of Al(OH)3 in the TC-1 model. ........................... 52

Establishment of T-lymphocytes quantification with triple-labeled

antibodies by flow cytometry in samples from patients of ARGOS

phase I clinical trial. ...................................................................... 53

Farmacometric study of nimotuzumab in colorectal cancer ........ 54

Pharmacophore modelling of PD-L1 immune checkpoint protein

through molecular dynamics simulations .................................... 55

Cervico-uterine cancer detection and prevention: next steps in the

era of precision medicine. ............................................................ 56

EGFR mutations as biomarker to target therapies in patients with

lung cancer in Cuba. ..................................................................... 57

HLA Polymorphism in Havana’s population. ................................ 58

Improved patent portfolio associated to the CK2-inhibitor peptide

CIGB-300 fostered by bioinformatics and OMICS ........................ 59

CIGB patent portfolio for cancer project research ....................... 60

1

OPENING SESSION

Thursday October, 24th (Room I)

20:00-20:15 Welcome remarks:

Dr. Luis Javier González (Center for Genetic

Engineering and Biotechnology, Havana, Cuba)

20:15-21:00 Opening lecture:

Dr. Gerardo Guillén (Director of Biomedical

Research at CIGB, Cuba)

21:00-21:45 Plenary lecture:

Dr. Patrick Aloy (Principal Investigator of the

Structural Bioinformatics Lab in the Institute for

Research in Biomedicine (IRB Barcelona), Spain)

“Extending the small molecule similarity principle to all

levels of biology”

21:45-22:45 “GET-TOGETHER WELLCOME COCKTAIL

2

ORAL PRESENTATIONS

Friday October, 25th

08:30-9:15 Plenary Lecture (Room I):

Benjamin Haibe-Kains, PhD (Canada)

“Biomarker Discovery in Cancer: Building a bridge between

preclinical and clinical research.”

“OMICs in Cancer” (Room I and Room II)

09:20-09:45 Ana C. de Carvalho, PhD (USA) (Room II)

“OMICS-credentialing of patient-derived glioblastoma

models for pre-clinical studies.”

09:50-10:15 Prof. Marcelo A. Soares (Brazil) (Room II)

“Novel gene targets in head-and-neck cancer revealed by co-

expression networks using multi-omic data integration.”

10:20-10:45 Prof. Patrick Aloy (Spain) (Room II)

“Personalized cancer therapy prioritization based on driver

co-occurrence patterns.”

10:50-11:15 Prof. Dimcho Bachvarov (Bulgaria) (Room II)

“Investigation of epithelial-mesenchymal transition

mechanisms implicated in epithelial ovarian cancer

dissemination by modulation of the LY75 gene expression.”

11:15-11:35

3

11:35-12:00 Alejandra Bernardini, PhD (Spain) (Room II)

“Molecular taxonomy of metastatic renal cell carcinoma and

its correlation with outcome to immunotherapy.”

12:05-12:30 Enzo Lalli, M.D. (France) (Room II)

“Steroidogenic Factor-1 is a Goldilocks transcription factor

regulating key genes implicated in tumorigenesis.”

12:35-13:00 Dr. Iraldo Bello (Cuba) (Room I)

“HeberFERON, combination of IFNs for cancer treatment.

The OMICS opportunities.”

13:05-13:30 Dr. Dania Marcia Vazquez-Blomquist (Cuba)

(Room I)

“RACE SEQ-MM: Predicting how any SNP of a patient can

affect endonucleotically active drugs of specific sequence,

such as antisense and siRNA.”

13:35-14:00 Dr. Vladimir Besada (Cuba)

“Phosphoproteomic study of the antitumorals

CIGB300/CX4945”

14:00-15:00

15:00-15:25 Bilateral meetings

4

Saturday October, 26th

08:30-9:15 Plenary Lecture (Room I):

Prof. Y-h. Taguchi (Japan)

“Application of tensor decomposition based unsupervised

feature extraction to multi-omics data set.”

“Novel strategies to overcome emerging cancer resistance

phenotypes ” (Room III)

Chairpersons: Dr. Michel Olin (USA) Dr. Mónica Bequet-Romero Ph.D

(CUBA)

09:20-09:45 Natasha Frank, MD (USA)

“Targeting therapeutic resistance in colorectal cancer”

09:50-10:15 Markus Frank, MD (USA)

“Role of ABCB5 in therapeutic resistance to targeted

melanoma therapies”

10:20-10:45 Dr. Sophie Tartare-Deckert (FRANCE)

“Microenvironmental influences in melanoma: a SPARC-ling

affair”

10:50-11:15 Dr. Marcel Deckert (FRANCE)

“Contribution of extracellular matrix signaling to melanoma

drug resistance”

11:15-11:35

5

“New targeted therapies and emerging profiling strategies in

cancer research.” (Room III)

Chairpersons: Dr Natasha Frank, MD (USA) Dr Marcel Deckert

(FRANCE)

11:35-12:00 Prof. Michael Olin (USA)

“CD200-Expresson on Vascular Endothelial cells is mediated

through VEGF”

12:05-12:30 Dr. Monica Bequet-Romero, PhD (CUBA)

“SaVax, a new VEGF active immunotherapy: Going for the

microenvironment”

12:35-13:00 Prof Silvio Perea (CUBA)

“CIGB-300: A Clinical-Stage anti-CK2 Peptide-based drug for

Cancer Therapy”

13:05-13:30 Ranjan J. Perera (USA)

“Integrated RNA and metabolite profiling of urine liquid

biopsies for prostate cancer biomarker and target discovery”

13:30-15:00

15:00-16:00 Posters discussion

16:00-17:00 Bilateral meetings

6

Sunday October, 27th

08:30-9:15 Plenary Lecture (Room I):

Prof. Harald Mischack (Germany)

“CE-MS complementarity to LC-MS: clinical proteomics on

oncology, kidney and cardiovascular diseases.”

“Original tumor targeting strategies (Room I)

Chairpersons: Prof. Ranjan J. Perera (USA), Dr Iraldo Bello Ph.D (CUBA)

09:20-09:45 Ana Campal (CUBA)

“Heberprovac, a GnRH based vaccine directed to prostate

Cancer. Clinical trials results and new challenges.”

09:50-10:15 Prof. Michael Olin (USA)

“Overpowering multiple inhibitory immune checkpoints with

a single peptide inhibitor.”

10:20-10:45 Dr. Julio Raul Fernández (CUBA)

“CRISPR/Cas9-mediated gene knockout of COMMD1

decreases sensitivity of H460 and HCT-116 cell lines to the

antitumoral peptide CIGB-552”

10:45-11:15 Closing remark, closing toast and official group

pictures

7

ORAL SESSION ABSTRACTS Extending the small molecule similarity principle to all levels of

biology Patrick Aloy

Principal Investigator of the Structural Bioinformatics Lab in the Institute for Research in Biomedicine (IRB Barcelona), Spain

paloy@irbbarcelona.org

Large scale small molecule bioactivity data are not routinely integrated in daily biological research to the extent of other ‘omics’ information. Compound data are scattered and diverse, making them inaccessible to most researchers and not suited to standard statistical analyses. In this talk, I will present the Chemical Checker (CC), a resource that provides processed, harmonized and integrated bioactivity data on small molecules. The CC divides data into five levels of increasing complexity, ranging from the chemical properties of compounds to their clinical outcomes. In between, it considers targets, off-targets, perturbed biological networks and several cell-based assays such as gene expression, growth inhibition and morphological profiles. In the CC, bioactivity data are expressed in a vector format, which naturally extends the notion of chemical similarity between compounds to similarities between bioactivity signatures of different kinds. I will show how CC signatures can boost the performance of drug discovery tasks that typically capitalize on chemical descriptors, including compound library optimization, target identification and anticipation of failures in clinical trials. Moreover, we demonstrated and experimentally validated that CC signatures can be used to reverse and mimic biological signatures of disease models and genetic perturbations, options that are otherwise impossible using chemical information alone.

8

Biomarker Discovery in Cancer: Building a bridge between

preclinical and clinical research Benjamin Haibe-Kains1,2,3,4

1Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada 2Department of Medical Biophysics, University of Toronto, Toronto,

Ontario, Canada 3Department of Computer Science, University of Toronto, Toronto, Ontario, Canada 4Ontario Institute for Cancer Research, Toronto, Ontario, Canada

5Vector Institute for Artificial Intelligence, Toronto, Ontario, Canada One of the main challenges in precision medicine consists of developing predictors of drug response to select the most beneficial therapy for each individual patient. In this context, preclinical models are crucial to study the association between molecular features of tumor cells and response to chemical perturbations. However, only few predictors have been successfully translated to clinical settings. Such a low success rate is due not only to the complexity of the mechanisms underlying anticancer drug response, but also to the lack of robustness of the predictors developed in preclinical settings. To address this issue we developed PharmacoGx, a computational platform enabling meta-analysis of large-scale drug screenings of in vitro and in vivo model systems, and PharmacoDB (pharmacodb.ca), a web-application enabling quick access to a large compendium of pharmacogenomics datasets. In this presentation I will show how we used our new platforms to develop univariate and multivariate predictors of drug response that can be further tested in clinical trial data.

9

Heberferon, combination of ifns for cancer treatment. The omics

opportunities. Bello-Rivero I1, Garcia-Vega Y2, Collazo-Caballero S3, Anasagasti-Angulo L4,

Valenzuela C2, Duncan-Roberts1 Y, Santana-Milian H5, Roben-Aguiar Y6, Martinez-Suárez C1, Acosta-Reyes O7, Nodarse-Cuni H1, Muzio-Gonzales V1.

1) Clinical Investigation Department, Center for Genetic Engineering and Biotechnology (CIGB); 2) Clinical Trial Department, Center for Molecular

Immunology, 3) Dermatology Department, Hermanos Ameijeiras Hospital; 4)Peripheral Tumor Department, National Institute of Oncology and Radiobiology;

5) Formulation Department, 6) Production Direction, 7) Business and Project Development, CIGB.

iraldo.bello@cigb.edu.cu HeberFERON is pharmaceutical formulation that combines in synergic proportions

IFNs α-g. The formulation reproduces several proprieties described for IFNS.

Pharmacological distinguishing property is the improved pharmacodynamics with

respect to separated IFNs, even PEGylated variants. Antitumor effectivity has been

proven in phase IV and real world treatment of patients with high risk and advanced

basal cell carcinoma with more that 60% of complete responses. However there is a

need for personalized use due to differences in clinical response rate depending on

tumor subtypes and likely patient characteristics. Patients with renal cell carcinoma

grade III or IV after nephrectomy greatly benefited from the parenteral

administration of HeberFERON with estimated overall survival of more than 50

months. The influence of tumor subtype and immunological patient’s environment

could dictate the final clinical outcome. While the origins of oncologic disease are

genetically encoded, the disease process is largely mediated through altered protein

function. Recent investigations suggest that each individual patient’s tumor

possesses unique kinase-driven cell signaling derangements, and that these

derangements derive, in part, from the tumor’s relationship with its host

microenvironment. Samples from more than 2000 patient with BCC treated with

HeberFERON, is an excellent and valuable opportunity for monitoring treatment

efficacy and toxicity and for predicting treatment outcome using OMICS approaches.

10

Omics approaches in glioblastoma multiforme under interferon co-

formulation treatment Vázquez-Blomquist D1, Besada V4, Miranda J2, Palomares CS5, Hardy A5, Baez S5, Ramos Y4, Guirola O5, Bringas R2, Leenstra S6, Wisniewski J7, Vonasek E8, Gil Y8,

Fernández de Cossío J2, Quiñones M4, Novoa LI1, Palenzuela D1, González LJ4, Bello I3 1Pharmacogenomic Group/System Biology Department, 2Bioinformatic Department,

3Clinical Assay Division, 4Proteomic Group/System Biology Department, 5 Bioinformatic Group/System Biology Department. Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba, 6Department of Neurosurgery, Erasmus MC, Rotterdam, the Netherlands, 7Protein Signaling and Proteomics, Max-Planck

Institute for Biochemistry, Munich, Germany. 8Instituto Venezolano de Investigaciones Científicas (IVIC), Venezuela.

dania.vazquez@cigb.edu.cu CIGB produces recombinant Interferons (IFNs) α and g and the co-formulation HeberFERON, with proved impact on non-melanoma skin carcinoma. In the last years, IFN combinations have also shown promising results in the aggressive and invasive brian tumor, Glioblastoma Multiforme (GBM). As HeberFERON could be used as a therapeutic for this tumor it would be interesting and necessary to understand the way it works in GBM and to obtain some predictive biomarkers for future clinical applications. To accomplish these objectives we used high throughput OMICs methodologies in two different models. We selected the very well-known U87MG cell line (TCGA molecularly as “classical like”) to perform Microarray (Illumina) and Proteomic (LC-MS/MS) experiments, using the same design, with HeberFERON treatment for 72h in comparison to treatments with IFNα or IFNg. We also carried out a Phosphoproteome experiment in this cell line treated with HeberFERON for 72h. A second approach took 34 GBM patient-derived clones, representing the four TCGA molecular classifications (15 classical, 4 proneural, 4 mesenchymal, 3 neural and 8 unclassified) which showed different proliferative responses to HeberFERON, independently of that classification. A Clariom S microarray experiment of these clones treated vs untreated with the co-formulation for 72h was also included as part of the data analysis. Additional validation by quantitative PCR was also included in the experimentation. It is our purpose to share the main OMICs results in both models in an individual but also integrative point of views. This analysis will guide us to describe a general mechanism of action for HeberFERON in GBM. The application of this product could be more precise based on that knowledge.

11

OMICS-credentialing of patient-derived glioblastoma models for

pre-clinical studies

deCarvalho AC1,2, Datta I3, Kim H4, Berezovsky A1,2, Nuga O1,2, Malta T1, Verhaak R4, Noushmehr H1, Poisson LM3

1) Dept. of Neurosurgery, Henry Ford Hospital, Detroit, MI USA; 2) School of Medicine, Wayne State University, Detroit MI USA; 3) Dept. of Public Health

Sciences, Henry Ford Hospital, Detroit, MI USA; 4) The Jackson Laboratory for Genomic Medicine, Farmington CT USA.

adecarv1@hfhs.org Glioblastoma is treated with maximal surgical removal, followed by radiation therapy

(RT) and DNA-alkylating agent temozolomide (TMZ). Despite this aggressive multi-

modality treatment, most patients relapse within months, with a dismal 2-year

survival rate of 15.2%. Small molecules and biologicals targeting multiple genomic

abnormalities and oncogenic signaling pathways in glioblastoma have largely failed

interventional clinical trials due to the lack of efficacy. The clinical translational value

of the use of patient-derived models in studies to uncover bona fide vulnerabilities

of cancer cells for targeted therapy depends on recapitulation of the genomic,

epigenomic and phenotypic heterogeneity of the original tumors. We will present

data credentialing a multiple model approach to studying therapeutic response in

glioblastoma through integrated multi-omics analysis. For a panel of molecularly

diverse glioblastoma tumors, whole genome copy number variation, exome

sequencing, DNA methylation, RNA sequencing and targeted proteomics were used

to compare multiple models with the original tumor, to investigate patterns of

molecular adaptive changes in the neoplastic cells to different selective pressures.

Taking advantage of the host uniformity of patient derived orthotopic mouse

xenograft models, we have identified tumor cell intrinsic transcriptional signatures of

fitness and response to DNA-targeting therapy. We further show that oncogene

amplifications in extra-chromosomal DNA is represented in the models and play a key

role in glioblastoma evolution and in therapeutic resistance.

12

Novel gene targets in head-and-neck cancer revealed by co-

expression networks using multi-omic data integration

Costa, RL1; Boroni, M2; Soares, MA1* 1Oncovirology Program, 2Bioinformatics Laboratory, Instituto Nacional do Câncer,

Rio de Janeiro, Brazil masoares@inca.gov.br

Human papillomavirus (HPV) is present in a significant fraction of head-and-neck squamous cell cancer (HNSCC). The main goal of this study was to identify distinct co-expression patterns between HPV+ and HPV− HNSCC and to provide insights into potential regulatory mechanisms/effects within the analyzed networks. We selected cases deposited in The Cancer Genome Atlas database comprising data of gene expression, methylation profiles and mutational patterns, in addition to clinical information. The intersection among differentially expressed and differentially methylated genes showed the negative correlations between the levels of methylation and expression, suggesting that these genes have their expression levels regulated by methylation alteration patterns in their promoter. Weighted correlation network analysis was used to identify co-expression modules and a systematic approach was applied to refine them and identify key regulatory elements integrating results from the other omics. Three distinct co-expression modules were associated with HPV status and molecular signatures. Validation using independent studies reporting biological experimental data converged for the most significant genes in all modules. This study provides insights into complex genetic and epigenetic particularities in the development and progression of HNSCC according to HPV status, and contribute to unveiling specific genes/pathways as novel therapeutic targets in HNSCC.

13

Personalized Cancer Therapy Prioritization Based on Driver

Alteration Co-occurrence Patterns

Patrick Aloy Principal Investigator of the Structural Bioinformatics Lab in the Institute for

Research in Biomedicine (IRB Barcelona), Spain paloy@irbbarcelona.org

Molecular profiling of personal cancer genomes, and the identification of actionable

vulnerabilities and drug-response biomarkers, are the basis of precision oncology.

Tumors often present several driver alterations that might be connected by cross-

talk and feedback mechanisms, making it difficult to mark single oncogenic variations

as reliable predictors of therapeutic outcome. In the current work, we uncover and

exploit driver alteration co-occurrence patterns from a recently published in vivo

screening in patient-derived xenografts (PDXs), including 187 tumors and 53 drugs.

For each treatment, we compare the mutational profiles of sensitive and resistant

PDXs to statistically define Driver Co-Occurrence (DCO) networks, which capture both

genomic structure and putative oncogenic synergy. We then use the DCO networks

to train classifiers that can prioritize, among the available options, the best possible

treatment for each tumor based on its oncogenomic profile. In a cross-validation

setting, our drug-response models are able to correctly predict 66% of sensitive and

77% of resistant drug-tumor pairs, based on tumor growth variation. Perhaps more

interesting, our models are applicable to several tumor types and drug classes for

which no biomarker has yet been described. Additionally, we experimentally

validated the performance of our models on 15 new tumor samples engrafted in

mice, achieving an overall accuracy of 75%. Finally, we adapted our strategy to derive

drug-response models from continuous clinical outcome measures, such as

progression free survival, which better represent the data acquired during routine

clinical practice and in clinical trials. We believe that the computational framework

presented here could be incorporated into the design of adaptive clinical trials,

revealing unexpected connections between oncogenic alterations and increasing the

clinical impact of genomic profiling.

14

Investigation of epithelial-to-mesenchymal transition mechanisms

implicated in ovarian cancer dissemination

Mehdi, S1,2, Bachvarova, M2, Scott-Boye,r M.P2, Droit, A1,2, Mcdonald, E3, Vanderhyden B3, Bachvarov, D1,2

1Dept. molecular medicine, Université Laval, Québec (Québec), Canada, 2Centre de recherche du CHU de Québec, Québec (Québec), Canada, 3Dept. molecular and

cellular medicine, University of Ottawa, Ottawa, ON, Canada. dimtcho.batchvarov@crhdq.ulaval.ca

The mechanisms for the tumorigenesis, progression and dissemination of epithelial

ovarian cancer (EOC) have not been yet fully clarified. We have recently shown that

the mannose receptor LY75 modulates epithelial-to-mesenchymal transition (EMT)

in EOC cells, as LY75 knockout (KO) induced mesenchymal-to-epithelial transition

(MET) in EOC cell lines with mesenchymal morphology, accompanied by a reduction

of their migratory and invasive capacity in vitro and enhanced tumor cell colonization

and metastatic growth in vivo. We used the Ly75-KO model to investigate for DNA

methylation alterations during EMT in EOC by applying the Reduced Representation

Bisulfite Sequencing (RRBS) technology. Numerous genes showing alterations in DNA

methylation during EMT were identified, which could represent new EOC therapeutic

targets. Consecutive methylation data analyses were indicative for the strong

implication of the Wnt/β-catenin signaling pathway in EMT-induced DNA methylation

alterations in EOC cells. This was indirectly confirmed by the identification of Ly75-

interacting partners using an immunoprecipitation technique, followed by mass

spectrometry. Moreover, we are currently applying the LY75 mediated-modulation

of EMT in EOC cells as a model to study in vivo the mechanisms of EMT implication in

EOC metastasis. We are using LY75-KO and control EOC cells to clarify the role of EMT

in EOC progression and treatment response in vivo in an intrabursal orthotopic

xenograft mouse EOC model.

15

Molecular taxonomy of metastatic renal cell carcinoma and its

correlation with outcome to immunotherapy

Bernardini A, Carril L, Suarez-Cabrera C, Dueñas M, Rodriguez Peralto JL, De Velasco G, Paramio JM.

There are three main histolopathological types of renal cell carcinomas (RCC): clear cell RCC –the most common type-, cromophobe RCC and papilar RCC. Furthermore, other RCC subtypes have been decrypted by mutational and transcriptomic data. The metastatic disease (mRCC) is frequently lethal due to its refractoriness to conventional chemotherapeutic agents. The application of targeted therapies and immune checkpoints inhibitors into the clinical of mRCC has increased the overall survival of RCC patients. The mRCC molecular subtypes are different in terms of prognosis but it is not clear how are related to therapy outcome: a variety of treatment responses is observed in mRCC patients subjected to 12 approved drugs belonging to 6 molecularly different mechanisms. We aimed to establish associations between transcriptomic patterns of mRCC patient samples and the response to immunotherapy. We included in the study mRCC samples from 80 patients that have been through at least two lines of treatment. Samples were histologically analyzed and classified into the main RCC types. Tissue samples with at least 60% of tumor content were selected and included in the study. We performed whole gene expression analysis of 45 RCC tumor samples using microarrays. Quality control and gene expression analysis were performed and it was possible to classify the samples in groups accordingly to patterns of gene expression and by hierarchical unsupervised classification independently of their histological type. We did comparisons between histological RCC types in terms of gene expression, gene set enrichment analysis and gene ontology pathways, finding different predictive responses to immunotherapy and the closest relationship between papilar and cromophobe types. We compared tumor transcriptomic features of responders and non-responders patients to the main drugs used in the set of patients: tyrosine kinase inhibitors and PDL1/PD1 immune checkpoint inhibitors, in order to look for transcriptomic profiles that differentiate response groups. The results of this study insight the increasingly importance of describing the complex molecular classification of RCC. This categorization contributes to define predictive biomarkers for current treatments of mRCC, and consequently, to stratify patients in order to provide the best possible treatment plan.

16

Steroidogenic Factor-1 is a Goldilocks transcription factor

regulating key genes implicated in tumorigenesis

LALLI, E Institut de Pharmacologie Moléculaire et Cellulaire; EXPOGEN-CANCER CNRS

International Associated Laboratory; Université Côte d'Azur, Sophia Antipolis, 06560 Valbonne, France

ninino@ipmc.cnrs.fr The transcription factor Steroidogenic Factor-1 (SF-1; NR5A1) regulates tissue-

specific gene expression in steroidogenic cells and has an essential role in the

development of adrenal glands and gonads. Furthermore, it is implicated in the

pathogenesis of adrenocortical tumors and its overexpression correlates with a

severe prognosis in adrenocortical carcinoma (ACC). We have shown that SF-1

regulates distinct set of genes in ACC cells according to its dosage. Those genes are

involved in defining several aspects of the malignant phenotype in ACC cells. We have

performed detailed kinetic analyses of the expression of genes regulated by SF-1 in

adrenocortical cancer cells. Our results reveal that different thresholds of gene

activation or repression exist according to the precise levels of SF-1 expression. We

could also ascertain that SF-1 autoregulates the expression of its own transcript, a

property that can be relevant to modulate its levels in ACC. The scenario then

emerges of SF-1 working as a Goldilocks transcription factor, with distinct dosages of

the protein each regulating a specific set of target genes. These properties are likely

to have an important role for transcriptional regulation of gene expression by SF-1 in

steroidogenic tissue development, physiology and disease. Further studies are in

progress with the aim to correlate SF-1 target gene expression to local chromatin

modifications and to perform single-cell gene expression analysis in conditions of

basal and increased SF-1 dosage in tissue culture cells and in a new mouse model of

Sf-1 overexpression.

17

Phosphoproteomic study of the antitumorals CIGB300 and CX-

4945.

Besada, V.1, Perera, Y.2, Ramos, Y.1, Perea, S.2, Guirola, O.3, Wisniewski, J.4, González, L.J.1

1Mass Spectrometry Laboratory, Department of Proteomics; 2Department of Pharmaceuticals, 3Group of Bioinformatics, CIGB, Havana; 4 Proteomics and Signal

Transduction, Max-Planck Institute for Biochemistry, Munich vladimir.besada@cigb.edu.cu

Casein kinase protein (CK2) is a ubiquitous enzyme that catalyze phosphorylation of

hundreds of substrates within cells. The enzyme is involved in cell growth,

proliferation and apoptosis, among others. CX-4945 is a small organic molecule that

binds to the catalytic cleft of casein kinase 2 alpha subunit impairing downstream

phosphorylation of hundreds of substrates. On the other side CIGB300 is a peptide

raised from a phage display library purposed to bind the phosphoaceptor substrate

target of the CK2. Both molecules are facing clinical trials as antitumoral drugs. A

phosphoproteomic study of both molecules was evaluated in two similar acute

myeloid leukemia cell lines: HL-60 and OCI-AML3. Here we present a preliminar

quantitative analysis.

18

Application of tensor decomposition based unsupervised feature

extraction to multi-omics data set

Taguchi, Y-h. Department of Physics, Chuo University, Tokyo 112-8551, Japan

tag@granular.com Although there have been numerous proposals on how to integrate multi-omics data

sets, most of them require the criteria on how to model the interaction between

individual omics data. In contrast to these so called “supervised methods”, the

proposed pethood is fully unsupervised and completely data driven method, which

is free from the bias introduced by human subjectivity. The proposed method can

achieve competitive performance with DIABLO proposed in mixOmics package in R.

In this lecture, In this lecture, I apply the proposed method to integrate, (A) mRNA

and miRNA expression of breast cancer [1], (B) multi-omics data taken from 26 non-

small cell lung cancer (NSCLC) cell lines [2], (C) gene expression and methylation taken

from brain of social insect [3], and (D) promoter methylation and miRNA expression

in TCGA data set [4]. The results are (A) treatment of multi classes (B) one-class

differential expression analysis (C) identification of genes that mediate social caste

development, (D) identification of correlation between regulation of genes by

promoter methylation and miRNA expression. Thus, the proposed method is

expected to have ability to integrate multi-omics data set successfully.

19

Targeting therapeutic resistance in colorectal cancer

Natasha Frank, MD Research Associate, Assistant Professor of Medicine, Harvard Medical School

Natasha.Frank@childrens.harvard.edu Colorectal cancer (CRC) is a leading cause of cancer-related deaths world-wide. While

early stages of colorectal cancer are highly curable by surgical resection, the

prognosis of patients with metastatic disease remains grave. Promising targeted

colorectal cancer therapies, including monoclonal antibodies against Epidermal

Growth Factor Receptor (EGFR), have resulted in significant improvement in overall

survival of patients with metastatic disease; however, the relatively transient or

individually restricted nature of clinical responses to currently available targeted

therapies underlines the urgency for further development of novel therapeutic

strategies that specifically target therapy-resistant cancer cell populations. ABCB5 is

a multidrug resistance mediator expressed in diverse human malignancies, including

CRC, where it mediates drug resistance to the chemotherapeutic agent 5-fluorouracil

(5-FU). In addition, recent studies revealed a novel drug efflux-independent role of

ABCB5 in CRC growth and aggressiveness through an anti-apoptotic mechanism

involving regulation of AXL, a receptor tyrosine kinase associated with adverse

prognosis in CRC patients that also represents a major mechanism of clinical

resistance to EGFR-targeting therapies. Here we will discuss our studies investigating

whether the ABCB5/AXL signaling pathway is responsible for emergence of resistance

to EGFR-targeting therapies in CRC, and whether specific targeting of this pathway

through monoclonal antibody-mediated ABCB5 blockade can improve clinical

outcomes in CRC patients with advanced disease.

20

Role of ABCB5 in therapeutic resistance to targeted melanoma

therapies

Markus Frank, MD Research Associate, Associate Professor of Pediatrics and Dermatology, Harvard

Medical School markus.frank@childrens.harvard.edu

ABCB5, a marker of immune-evasive cancer stem cells, is functionally required for

tumor initiation and metastatic progression in human malignant melanoma and

additional human malignancies. While its function in chemotherapeutic cancer

multidrug resistance has been broadly established, the potential role of ABCB5 in the

resistance to novel targeted melanoma therapies remains largely unknown. Here we

show in novel in vivo models of melanoma in which ABCB5 has been genetically

ablated a critical role of the molecule in mediating therapeutic resistance to the BRAF

inhibitor vemurafenib. Our findings suggest that ABCB5-expressing melanoma

initiating cells represent a malignant cell subset that exhibits resistance to both

immune- as well as gene-targeted therapies and that this broad resistance pattern

can be reversed through ABCB5-inhibitory therapeutic strategies.

21

Microenvironmental influences in melanoma: a SPARC-ling affair

Dr. Sophie Tartare-Deckert University Côte d'Azur, Mediterranean Center for Molecular Medecine (C3M) /

Inserm U1065, Team Microenvironment, Signaling and Cancer, Nice, France. Sophie.Tartare-Deckert@unice.fr

Our laboratory is interested in understanding microenvironmental influences and

signaling networks that drive tumor growth and dissemination. We have been

particularly involved in studying the role of tumor microenvironment in metastatic

niche formation and response to therapies in melanoma, the most aggressive and

lethal form of skin cancers. The matricellular protein SPARC (Secreted Protein, Acidic

and Rich in Cysteine) is an important driver of theses processes, particularly by

inducing tumor cell invasion and p53-dependent survival, and its expression is linked

to an aggressive mesenchymal phenotype. We recently revealed that SPARC also

functions as a critical tumor-secreted permeability factor in the extravasation stage

of the metastatic cascade. Our current work focuses in deciphering how tumor-

secreted factors and matrix environmental properties influence the invasive

melanoma cell phenotype and the formation of the lymph node pre-metastatic niche.

I will present an overview of our data supporting a role for SPARC in melanoma cell

phenotypic plasticity, metastasis and resistance to therapies targeting the BRAF

oncogenic pathway. Exploiting the pathological expression and function of SPARC in

melanoma is emerging as a promising strategy to develop new biomarker and

therapeutic tools for clinical management of the metastatic and treatment-refractory

disease.

22

Contribution of extracellular matrix signaling to melanoma drug

resistance

Marcel Deckert University Côte d'Azur, Mediterranean Center for Molecular Medecine (C3M) /

Inserm U1065, Team Microenvironment, Signaling and Cancer, Nice, France. Marcel.Deckert@unice.fr

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark

of several solid cancers and is associated with therapy failure. BRAF-mutant

melanomas treated with MAPK inhibitors (MAPKi) almost invariably develop

resistance that is frequently associated with transcriptional reprogramming and a

dedifferentiation cell state. Melanoma cells produce their own ECM, an event that is

promoted by MAPKi. Yet, the contribution of cancer cell-derived ECM and tumor

mechanics to drug adaptation and therapy resistance remains poorly understood.

Recent results from our lab reveal that melanoma cells can adapt to targeted

therapies through a mechanosignaling loop involving the autocrine remodeling of a

drug-protective ECM. I will present results showing that therapy resistant cells

associated with a mesenchymal de-differentiated state display elevated

responsiveness to collagen stiffening and force-mediated ECM remodeling through

activation of actin-dependent mechanosensors YAP and MRTF. Short-term MAPKi

exposure of melanoma cells also induces mechanosignaling associated with

deposition and remodeling of a fibrillar ECM. This provides a favored matrix

environment to promote tolerance to MAPKi in a YAP and MRTF-dependent manner.

Collagen remodeling and tumor stiffening are also observed in vivo upon exposure of

BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPKi.

Importantly, pharmacological inhibition of YAP markedly decreases treatment-

induced excessive collagen deposition, leading to normalization of tumor ECM and

enhancement of BRAF inhibitor efficacy. Finally, I will present preliminary data

showing how we can therapeutically manipulate ECM-mediated signaling via the two

non-integrin tyrosine kinase receptors for collagens DDR1/2 to overcome melanoma

drug resistance.

23

CD200-Expresson on Vascular Endothelial cells is mediated through

VEGF

Dr. Michael Olin, PhD Assistant Professor in the Division of Pediatric Hematology and Oncology

University of Minnesota. olin0012@umn.edu

Central nervous system tumors are the number one killer of children with cancer.

Among those, glioblastoma multiforme (GBM) is an incurable primary brain tumor.

The standard of care consists of resection followed by radiation and chemotherapy

using temozolomide and is associated with a median overall survival of 14.6 months.

To overcome this dismal outcome, clinicians are turning to immunotherapy

approaches, which have demonstrated promising results in other solid tumors.

However, immunosuppression by the tumor microenvironment prohibits a durable

anti-tumor response and the optimization of immunotherapy, especially in GBM.

Tumors have capitalized on immune regulatory mechanisms that facilitate the

inhibition of an immune response. The CD200 checkpoint includes the tumor-bound

protein CD200 and its inhibitory receptor. We recently reported that CD200 is also

expressed on tumor vascular endothelial cells and that this expression is upregulated

by VEGF, creating an immunological barrier around the tumor microenvironment.

However, as extracellular proteases are abundant in the sera, it is highly probable for

the soluble CD200 to be degraded. Our hypothesis is that tumor-derived VEGF+

extracellular vesicles modulate CD200-expression on tumor vascular endothelial

cells. There is abundant evidence in the literature that extracellular vesicles derived

from tumors suppress antigen-specific and non-specific anti-tumor responses,

mediating a broad array of detrimental effects on the immune system. We now know

that tumor-derived extracellular vesicles contain CD200 and VEGF. In addition, others

have reported that miR-150, which plays a role in tumorigenesis, interacts with TAMs

to induce VEGF, suggesting a link between tumor-derived extracellular vesicles, and

the upregulation of C200. We propose i) CD200-expressing tumor-derived

extracellular vesicles induce immune suppression, ii) VEGFpos-tEVs upregulates

CD200 on vascular endothelial cells and/or iii) miR150 interaction with tumor

associated macrophage upregulate CD200 on vascular endothelial cells maintain an

immune suppressive tumor microenvironment.

24

CIGB-247, a VEGF based active immunotherapy: Going for tumor

microenvironment

Bequet-Romero, M1; Canaan-Haden C1, Rodríguez L, Rodríguez S1, González-Moya I1,

Etchegoyen AY 1, Sánchez, J1; Morera, Y1; Ayala, M1. 1 Department of Pharmaceuticals, Center for Genetic Engineering and

Biotechnology, Havana City, Cuba monica.bequet@cigb.edu.cu

Cancer vaccine effects are often hindered by the strong immunosuppressive effect of the tumor microenvironment that neutralizes the effector cells of the immune system at the tumor site. The Vascular Endothelial Growth Factor (VEGF) known also by its association to a bad prognosis in cancer patients is a key molecule in these processes. CIGB-247 is an active immunotherapeutic approach for cancer treatment that targets VEGF by inducing specific humoral and cellular responses towards the autologous protein in the systemic compartments of several species including patients with advanced solid tumors. Such responses are obtained after the administration of a non-active variant of VEGF in the context of adjuvants like Aluminum Phosphate or the Very Small Size Particles from Neisseria Meningitides membrane that incorporates the synthetic ganglioside N-Acetil GM3 (sVSSP). Herein the first studies aiming to characterize CIGB-247 effects on the tumor microenvironment in heterotopic CT26 tumors implanted in BALB/c mice will be presented and discussed. Blocking of VEGF binding to VEGFR2 was demonstrated in the tumor compartment and a relevant increase on the number of T cell clones secreting IFN-γ in response to VEGF was detected in tumor draining lymph nodes for CIGB-247 receiving animals. These mice also exhibited a significant reduction on protein concentration of VEGF, PlGF, GCSF and CXCL1 with an induction of IFN-γ, IL-2 and GRZ-B in the tumor microenvironment indicating that some known resistance mechanisms for other antiangiogenics might not be in place for the strategy under evaluation. Flow cytometric evaluation of dissociated tumor tissue, spleen, lymph nodes and whole blood indicate that the immunotherapy effectively increased in both the tumor and the draining lymph nodes the number of CD4+ and CD8+, as well as the activation marker CD69 among them, while exposition of the senescence related molecules PD1 and VEGFR2 was significantly reduced. Such effects were related to the reduction of tumor sizes. Altogether, our results indicate that HeberSaVax administration induces a specific immune response to VEGF that can be detected within the tumor compartment where the treatment is able to reduce pro-angiogenic protein expression and induces CD8 effector cytokines in parallel to an improvement towards reactivity of the infiltrating leucocytes.

25

CIGB-300: A Clinical-stage Anti-CK2 Peptide-based Drug for Cancer

Therapy Prof Silvio E. Perea, PhD

Head of the Molecular Oncology Laboratory at the Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Head of the Cancer Council at the CIGB.

silvio.perera@cigb.edu.cu

CIGB-300 is a first-in-class synthetic peptide initially discovered by screening a

peptide phage display library using a CK2 phosphoaceptor site as target. Previous in

vivo findings had indicated that CIGB-300 preferentially binds to B23/NPM protein

and inhibits its CK2-mediated phosphorylation leading to apoptosis in solid tumor cell

lines. However, in vitro assays with recombinant CK2 along with in vivo

phosphoproteomic studies in lung cancer and AML cells, have suggested that CIGB-

300 impairs the CK2-mediated phosphorylation in a multitarget fashion. Accordingly,

beside the proapoptotic effect, CIGB-300 has also showed both antiangiogenic and

antimetastasic activity in animal models when administered by systemic routes. At

present, CIGB-300 is being explored as a therapeutic candidate and data from

different Phase 1 clinical studies have already shown safety, tolerability,

pharmacokinetic and clinical activity using either intralesional or systemic

administration. Also, in a dose-finding Phase 2 study, CIGB-300 concomitantly

injected with chemoradiotherapy in cervical cancer exhibited higher complete

response rate than chemoradiotherapy alone in a particular dose range. Importantly,

clinical data on CIGB-300 provide useful clues for designing upper Phase trials in

cervical cancer patients. Collectively, our data outline important new clinical and non-

clinical insights that reinforce CIGB-300 as a novel peptide-based candidate with

perspectives to treat cancer.

26

CE-MS in clinical proteomics: application in oncology, kidney and

cardiovascular diseases Harald Mischak

Mosaiques Diagnostics, Germany, and University of Glasgow, United Kingdom In depth investigation has demonstrated that naturally occurring peptides demonstrate significant association with several pathologies. These peptides represent excellent biomarkers for specific diseases and have the potential of mirroring biology/pathophysiology. Among the technologies employed to analyze peptides in depth, capillary electrophoresis coupled mass spectrometry (CE-MS) has established itself as a leading technology, due to its robustness and reproducibility. In direct comparison, complementarity of CE- and LC-MS becomes evident, due to the different separation principles. Urine has emerged as a prime source for proteomic biomarkers, also due to its ease of collection. Urinary peptides are produced either by the kidney, or a result of plasma filtration. To date, over 100000 peptides have been tentatively identified in urine, over 5000 are sequenced. Large multicenter studies involving over thousand individuals indicate a significant added benefit of urinary proteomic biomarkers. More than 70.000 samples have been analyzed to date in a comparative way using CE-MS, enabling the generation of the worldwide by far largest peptidome database that serves as basis for the identification of biomarkers based on large numbers of independent samples, a prerequisite when aiming to identify valid biomarkers. The results from these studies also demonstrated that single biomarkers are of limited value as a result of moderate specificity and high variability. However, combining multiple biomarkers into specific classifiers has presented itself as an the ideal approach to assess disease in a non-invasive way with high precision. Since more that two thirds of the urine peptides appear to originate in the kidney, an obvious target for urinary clinical proteomics is the application in chronic kidney disease (CKD). CKD is a major health burden with associated costs exceeding 100 bio € per year worldwide. Urinary peptide biomarkers enable early and accurate detection of CKD (2-5 years prior to clinical diagnosis) and prognosis of future development. Specific urine collagen fragments show the highest predictive value at very early stage CKD. This opens a therapeutic window at an early point in time, possibly even enabling curative treatment. The results have led to the initiation of a first large, multicentric, randomised controlled clinical trial, PRIORITY, aiming at preventing the development of diabetes-associated CKD by early intervention guided by urinary proteomics. Additional fields of clinical application are in the (early) detection of cardiovascular diseases (heart failure, and coronary artery disease) and in the detection and, in some cases, monitoring of malignancies like prostate or bladder carcinoma.

27

Heberprovac, a GnRH based vaccine directed to prostate Cancer.

Clinical trials results and new challenges. Jesús A. Junco1, Ranfis Rodríguez4, Franklin Fuentes1, Idania Baladrón2, Lesvia

Calzada1, Carmen Valenzuela5, Eddy Bover1, Eulogio Pimentel2, Roberto Basulto1, Maria D. Castro1, Francisco Sariol3, Ayni Rodríguez6, Hilda Garay2, Osvaldo Reyes2,

Matilde López2, Ana Campal1, Verena Muzio2, Gerardo Guillén2. 1. Centro de Ingeniería Genética y Biotecnología de Camaguey. Ave Finlay y

Circunvalación Norte, CP 70100, Camaguey, Cuba. 2. Centro de Ingeniería Genética y Biotecnología. Ave 31 e/ 158 y 190, Playa, P.O. Box 6162. Habana 10600, Cuba. 3. Hospital Oncológico Provincial Marie Curie. Carretera Central Oeste esquina

Madame Curie. CP. 70700, Camaguey, Cuba. 4. Instituto Nacional de Oncología y Radiobiología (INOR) Calle 29 esq. a F. Vedado, CP. 1040. La Habana, Cuba.

5. Centro de Inmunología Molecular, Calle 15 esq. 216 S/N, Siboney, Playa, A.P 16040, La Habana, Cuba. 6. Universidad Médica de Camaguey. Carretera Central

Oeste entre Madame Curie y Calle 9. Camagüey. CP 70100. Camaguey, Cuba jesus.junco@cigb.edu.cu

GnRH-based vaccines represent a promising anti-hormonal treatment alternative in prostate cancer, because they can reduce serum testosterone to castrating levels, avoid the "hot flushes” produced by GnRH analogues and can be administered in acute and complicated forms of prostate cancer. The present study assesses the application of Heberprovac, a GnRH based vaccine candidate for patients suffering from advanced prostate cancer and their following up to ten years in 2 clinical trials. The main objective of the first clinical trial was to evaluate the safety and possible efficacy indicators and the second, Phase II clinical trial, to evaluate the safety and efficacy of this vaccine candidate in 4 levels of dosage. To this aim, 6 patients affected by advanced prostate cancer diagnosed by biopsy, were included in the first clinical trial and 56 were treated in the second one. As result of the first clinical trial it was generated a 100% of effective anti GnRH immune response that corresponded with a significant testosterone reduction until castration levels, normalization of PSA and full clinical response in the whole patients subset. On the other hand, the Phase II clinical trial revealed that after the first immunizations, the patients exhibited anti GnRH antibodies and in turn, testosterone levels reduction. In concordance with the hormonal and immunological response, the patients exhibit a decrease of both; the number of obstructive symptoms as well as the severity of them. There was also a normalization of the prostatic specific antigen (PSA) in the 80% of the patients after they finished the last immunization and the clinical evaluation demonstrated the significant reduction of the primary tumor from grades III/IV to I/II in all the patients that respond biochemically. Currently a Phase II/III controlled clinical trial is planned to be carried out in more than 300 advanced prostate cancer patients in Cuba.

28

Overpowering multiple inhibitory immune checkpoints with a

single peptide inhibitor. Dr. Michael Olin, PhD

Assistant Professor in the Division of Pediatric Hematology and Oncology University of Minnesota.

olin0012@umn.edu Given the modest effects current immune checkpoint-inhibitors have on patients

with CNS tumors, we focused our studies on the novel CD200 immune checkpoint.

The CD200 checkpoint suppresses the immune system through binding of the

inhibitory CD200 protein to the inhibitory receptor (CD200R1) expressed on immune

cells. In addition to CD200R1, the CD200 checkpoint includes activation receptors

(CD200AR), which we are targeting with a peptide ligand (CD200AR-L) to reverse the

inhibitory effects of the CD200 protein. We showed that spontaneous high-grade

glioma dogs receiving a canine specific CD200AR-L in combination with autologous

tumor lysate vaccine had an increased median overall survival of 9 months, compared

to 6.3 months with lysate alone, resulting in an overall 36-month survival of 21%.

However, we have a 45% event-free (progression-free) group of dogs that died of

non-tumor related deaths with an extended median survival of 18 months. Six dogs

remain on trial, 87% of the dogs receiving tumor lysate only died of tumor recurrence

within 6 months. Serum chemistry profiles and physical examinations showed that

the peptide did not induce any systemic toxicity. To determine how we obtained

these unprecedented results, we focused on the mechanism of CD200AR-L/CD200AR

binding. We discovered DAP10/12 signaling pathways and CD200ARs are

upregulated, while the inhibitory CD200R1 is downregulated. We discovered the

molecular connection between CD200, PD-1 and CTLA4. We demonstrated that our

murine and human CD200AR-L downregulates the inhibitory PD-1 and PD-L1 and

inhibits the upregulation of CTLA4. Therefore, we attribute the success of CD200AR-

L to the ability to concurrently modulate multiple immune checkpoints. We have now

developed human GMP grade CD200AR-L peptides to develop an immunotherapy

approach for patients with CNS tumors; we will conduct a Phase I trial utilizing the

peptide ligand concomitantly with an allogeneic GBM6-AD cell line as a tumor vaccine

and the adjuvant imiquimod.

29

CRISPR/Cas9-mediated gene knockout of COMMD1 decreases

sensitivity of H460 and HCT-116 cell lines to the antitumoral

peptide CIGB-552. Garcia G, Fernández JR; Palenzuela D, Guerra M.

Centro de Ingeniería Genética y Biotecnología (CIGB), La Habana, Cuba julio.fernandez@cigb.edu.cu

Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a critical role across

many cellular processes including embryonic and neuronal development, cell

proliferation, apoptosis, immune responses to infection, and inflammation.

Dysregulation of NF-κB signaling is associated with inflammatory diseases and certain

cancers. Constitutive activation of NF-κB signaling has been found in some types of

tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic

blockade of NF-κB signaling in cancer cells provides an attractive strategy for the

development of anticancer drugs. CIGB-552 is a novel synthetic peptide derived by

modification of its primary structure from the antimicrobial peptide LALF32-51

(Limulus sp) which has been shown to be a potential candidate for the anticancer

therapy and one of its useful property is the cell-penetrating capacity. CIGB-552 binds

and stabilizes COMMD1 protein, which inhibits the transcription factor NF-kB, a

factor linked to the proliferation, survival and metastatic capacity of the cancer cell.

The accumulation of COMMD1 promotes the ubiquitination of the RelA subunit of

NF-kB and its proteosomal degradation, causing apoptosis of the cancer cells. CIGB-

552 modulates the expression of genes related to the cell cycle, apoptosis and

oxidative stress among others. CIGB-552 peptide blocks cell proliferation followed by

death by apoptosis and causes oxidative damage to lipids and proteins. Here, we

provided the expression profile data of COMMD1 knockout in a H460 cell line treated

with CIGB-552 and shows that the Knockout of COMMD1 decreases sensitivity of

H460 and HCT-116 cell lines to the antitumoral peptide CIGB-552.

30

31

POSTER SESSION CANCER 01 A co-formulation of interferons type I and II enhances

temozolamide response in glioblastoma by decreasing MGMT levels Dania Vázquez-Blomquist1, Sieger Leenstra*, Jan-willem Jachtenberg*, Marielle van der Kaaj*, Adelaida Villarreal1, Iraldo Bello-Rivero2.

CANCER 02 Global analysis of genes expression in human glioblastoma derived clones treated with the interferon co-formulation HeberFERON. Vázquez-Blomquist D1, Pérez W1, Villareal A1, Palenzuela D1, Bello I2.

CANCER 03 First exploration of proteomic profile regulated by the co-formulation of type I and II interferons HeberFERON Vázquez-Blomquist D1, Besada V2, Miranda J4, Ramos Y2, Palomares CS3, Guirola O3, Vonasek E6, Gil Y6, Díaz T1, Quiñones M2, Bringas R4, González LJ2, Bello I5.

CANCER 04 Clinical development of CIGB-247 active cancer immunotherapy: Entering Phase II/III clinical trials Hernández-Bernal F,. Martin Y, Catasus K, de la Torre A, Selman-Housein KH, Baladrón I, Perera JC, 1Sánchez-Ramírez J, 1Morera Y, Gavilondo JV, Ayala M, and Bequet-Romero M.

CANCER 05 Where VEGF should be measured? An experience obtained from phase I clinical trials cancer patients treated with a VEGF vaccine. Sánchez, J1; Bequet-Romero, M1; Morera, Y1; Hernández, F2; de la Torre, A3; Selman-Housein, KH4; Martín, Y2; Limonta, M1; Ayala, M1.

CANCER 06 Antitumoral effects of a VEGF based active immunotherapy formulated in Alum Phosphate in orthotopic ovarian tumors in C57Bl/6 mice. I. González-Moya1, C. Canaan-Haden1, S. Rodríguez1, AY Etchegoyen1, D. Pichardo2, A. Blanco2, J. Sánchez Ramírez1, and M. Bequet-Romero1

32

CANCER 07 Transcriptional evaluation of genes related to the potential mechanism of action of the VEGF based active immunotherapy CIGB247, in the context of CT26 ectopic tumors: Preliminary results. 1Rodríguez S, 2Bakos L., 1 Canaan-Haden C, 1González-Moya I., 1Sánchez-Ramírez J., 1Morera Y., 2Palenzuela D., and 1Bequet-Romero M.

CANCER 08 ELISA method for quantification of antibodies against VEGF protein. Coizeau, E1; Avila, L1; Chávez, S1; Freyre, G1;Vázquez, A1; Sánchez, J2; Bequet-Romero, M2 and Lemos, G1.

CANCER 09 Clinical Research of CIGB-300 in patients with Locally Advanced Cervical Cancer I Baladrón, AM Solares, M Sarduy, G Crespo, LA Estévez, D González, A Santana, JL Soriano, N Batista, A Lopez, Y Perera, H Farina, C Valenzuela, P López, Ignacio Hernández, Idrián García, L Gonzalez, A Palau, I Hernández, I García, A Díaz, C López, SE Perea, V Muzio.

CANCER 10 Effect of the clinical-grade Casein Kinase 2 inhibitor CIGB-300 on NF-kB signaling in Acute Myeloid Leukemia cells Ramon AC*, Vázquez-Blomquist D, Besada V, Rosales M, Wisniewski J, González L.J, Perera Y, Perea SE.

CANCER 11 NPM1c+ as a putative novel target of interaction for the antitumoral peptide CIGB-300 Pérez GV1*, Ramon AC1, Rosales M1,2, Perea SE1 and Perera Y1

CANCER 12 Modulation of PD-L1 expression by CIGB-300 in human cervical cancer cell lines Aguilar-Noriega D, Hurtado D, Vazquez D, Perea SE.

CANCER 13 MultiOMICs data analysis enables identification of biological processes and pathways modulated by CIGB-300 in leukemia cells Rosales M *, Vázquez-Blomquist D, Besada V, Ramón AC, Perera Y2, Perea SE

33

CANCER 14 Phosphoproteomic profile modulated by the anticancer peptide CIGB-300 in human promyelocytic leukemia (HL-60) cells. Rodríguez-Ulloa, A.(1)*; Perera, Y.(1); Ramos-Gómez, Y.(1); González, L.J.(1); Wisniewski, J.(2); Perea, S.E.(1); Besada, V.(1)

CANCER 15 The influence of different peptide to increase the immunogenicity of the GnRH vaccine for prostate cancer treatment. Fuentes F, Junco J, Calzada L, Bover E, Serradelo JA, Hernández E, Pimentel E, Basulto R, Reyes O, Garay H and Guillen G

CANCER 16 Bioequivalence of natural and synthetic VSSP using the peptide GnRHm1/TT for the treatment of prostate cancer. Calzada L, Fuentes F, Junco J, Campal A, Garay H, Serradelo A, Lopez E and Guillén G

CANCER 17 Establishment of an ELISA for the quantification of the CIGB-552 peptide in plasma samples from patients of ARGOS Phase I Clinical Trial. Vázquez, A1; Sobrino, X1; Cabrales, A1; Támbara, Y1; Guerra, M2 and Lemos, G1.

CANCER 18 Studies of different doses of the CIGB550-E7 vaccine candidate with a fixed dose of Al(OH)3 in the TC-1 model. Rodríguez N, Alfonso AB, Granadillo M, Batte A, Torrens I

CANCER 19 Establishment of T-lymphocytes quantification with triple-labeled antibodies by flow cytometry in samples from patients of ARGOS phase I clinical trial. Avila L1, Vázquez A1, Freyre F1, Martinez R1, Guerra M2 and Lemos, G1.

CANCER 20 Farmacometric study of nimotuzumab in colorectal cancer Fernández B1, Rodríguez L2, de Castro N2, Lavastida A4

CANCER 21 Pharmacophore modelling of PD-L1 immune checkpoint protein through molecular dynamics simulations Montes, L.; Mejías, C.; Guirola, O.

CANCER 22 Cervico-uterine cancer detection and prevention: next steps in the era of precision medicine. González, N; Díaz, M; García, G; Ortega, A.

34

CANCER 23 EGFR mutations as biomarker to target therapies in patients with lung cancer in Cuba. Ortega A, Garcia G, Hernández B

CANCER 24 HLA Polymorphism in Havana’s population. Chang, A; Marcell, L; Rodríguez E; Figueras JP; Guevara, LM; Valdés, R; Ustariz, CR.

CANCER 25 Improved patent portfolio associated to the CK2-inhibitor peptide CIGB-300 fostered by bioinformatics and OMICS Gonzalez, Sa; Perea, Sb; Perera, Yb; Rodriguez, Ac; Besada, Vc; Gonzalez, LJc; Rodriguez, Rc; Mazola, Yc; Musacchio, Ac; Vazquez, Ma; Ubieta, Ra.

CANCER 26 CIGB patent portfolio for cancer project research Vazquez M.1, Gonzalez S.1, Ubieta R.1 , Perea S.2 , Rodriguez R.2, Bequet-Romero M.2, Abrahantes M.C3, Bello I.4, Guerra M.2, Torrens I.2, Junco J.2, Chinea G.2, Lamdan H.2

35

POSTER SESSION ABSTRACTS A co-formulation of interferons type I and II enhances

temozolamide response in glioblastoma by decreasing MGMT

levels Dania Vázquez-Blomquist1, Sieger Leenstra*, Jan-willem Jachtenberg*, Marielle van

der Kaaj*, Adelaida Villarreal1, Iraldo Bello-Rivero2. 1. Pharmacogenomic Group, Biomedical Research Direction and 2. Clinical Assay

Direction, Center for Genetic Engineering and Biotechnology, Havana, Cuba. *Erasmus MC, Neurosurgical Department, Rotterdam, The Netherlands.

dania.vazquez@cigb.edu.cu

Temozolomide (TMZ) is a commonly chemotherapeutic used in the treatment of glioblastoma. The MGMT repair enzyme (O´ (6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response and Interferons (IFNs) type I can downregulate MGMT gene and protein expressions improving survival in patients with unmethylated MGMT promoter. HeberFERON is a new co-formulation of IFNs type I and II with higher antiproliferative effect than individual IFNs over glioblastoma cell lines. The objective of this work was to investigate the proliferative response to HeberFERON and its combination with TMZ in patient-derived glioblastoma cultures, in relation to MGMT promoter methylation, and also to investigate the regulation of MGMT gene expression after the treatment with HeberFERON by quantitative PCR. We showed that response of glioblastoma clones to HeberFERON is variable, dose-dependent and apparently independent from MGMT promoter methylation status and TCGA classification. When we combined HeberFERON with TMZ there was an increase in cell death for those clones with unmethylated MGMT promoter, probably due to the decrease or maintainance of MGMT gene levels. We also showed a preliminary association between MGMT and P53 levels. The relevance of these biomarkers should be address in the future but the combination of HeberFERON with TMZ could have advantages for the treatment of glioblastoma with MGMT unmethylated promoter status.

36

Global analysis of genes expression in human glioblastoma

derived clones treated with the interferon co-formulation

HeberFERON.

Vázquez-Blomquist D1, Pérez W1, Villareal A1, Palenzuela D1, Bello I2. 1Pharmacogenomic Group, Biomedical Research Direction and 2Clinical Assay Direction, Center for Genetic Engineering and Biotechnology, Havana, Cuba.

dania.vazquez@cigb.edu.cu

Glioblastoma multiform (GBM) are aggressive brain tumors, with a median survival of 12-14 months. Despite aggressive therapeutic regimens, patients suffer recurrence due to molecular heterogeneity of GBM; few biomarkers with prognosis and predictive values have been described. CIGB produces recombinant Interferons (IFNs)

and and the co-formulation HeberFERON. HeberFERON have shown superior antiproliferative effect over GBM cell lines compared to individual IFNs; in a compassionate study in humans it improved patients´s quality of life and the median survival compared to historical data. We previously tested the growth inhibitory response of 34 patient-derived GBM clones, with different molecular profiles, to HeberFERON. We found the inhibitory response was independent of TCGA molecular classification and subdivided the clones in high (H), low (L) and intermediate (I) responders in relation to their cell growth inhibitory response. Total RNAs from each clone untreated or treated with HeberFERON for 72h were used to investigate the gene expression profile by Clariom S microarray (Affymetrix, Hamilton Health Sciences Corp., Canada). Analysis of gene expression data using functional analysis and network interfunctional connections tools were used to explore the mechanisms of action and possible predictive biomarkers. Contrasts between all treated vs untreated clones and H vs L responders showed biological processes&pathways related to IFN signaling, MHC peptide presentation, translation, RNA processing, ubiquitin proteosome pathway and energy related metabolic processes. Although differences in response of H vs L in “classical subtype” clones can be explained in this way, this looks less probable for “proneural subtype” clones. Quantitative PCR assays confirmed some biomarkers. These preliminary results could have an impact in HeberFERON use in GBM.

37

First exploration of proteomic profile regulated by the co-

formulation of type I and II interferons HeberFERON Vázquez-Blomquist D1, Besada V2, Miranda J4, Ramos Y2, Palomares CS3, Guirola O3,

Vonasek E6, Gil Y6, Díaz T1, Quiñones M2, Bringas R4, González LJ2, Bello I5. 1Pharmacogenomic Group, 2Proteomic Group and 3Bioinformatic Group in System Biology Department, 4Bioinformatic Department, 5Clinical Assay Direction. Center

for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.6Instituto Venezolano de Investigaciones Científicas (IVIC), Venezuela.

dania.vazquez@cigb.edu.cu

CIGB produces recombinant Interferons (IFNs) α and and a synergic co-formulation, HeberFERON. HeberFERON have shown a high impact over non-melanoma skin carcinoma and, recently, encouraging results over Glioblastoma Multiforme (GBM)-derived cell lines and patients in a compassionate study. GBM-bearing patients display a short overall life expectancy and the occurrence of recurrences after the most challenging treatments. HeberFERON can become in a new option for these patients, therefore the understanding of its mechanism of action could be very useful for future clinical applications. We selected as a model the cell line U87MG and performed an experiment for 72h with three replicates per treatment: untreated, treated with IFNα or IFN and treated with HeberFERON. A proteomic experiment using LC-MS/MS (IVIC, Venezuela) was performed and lists of proteins differentially expressed from each treatment group, compared with the untreated, were generated. We analyzed the data in order to understand the mechanism of action of HeberFERON over U87MG and what makes it different from its individual IFNs components. Different functional classification and enrichment analysis, network connections and on-net analysis were carried out. The existence of a “mirror” microarray experiment, with same design, allowed us to contrast gene and protein expressions with HeberFERON for a better interpretation of overall results. The identification of mechanism-related biomarkers and biomarkers to predict the response to HeberFERON would be very valuable for a more precise application in GBM.

38

Clinical development of CIGB-247 active cancer

immunotherapy: Entering Phase II/III clinical trials

1Hernández-Bernal F,. 1Martin Y, 1Catasus K, de la Torre A, Selman-Housein KH, 1Baladrón I, Perera JC, 1Sánchez-Ramírez J, 1Morera Y, 1Gavilondo JV, 1Ayala M, and

1Bequet-Romero M. Center for Genetic Engineering and Biotechnology, Center of Medical and Surgical

Research (CIMEQ) Havana, Celestino Hernández hospitalCuba. monica.bequet@cigb.edu.cu

CIGB-247 is a cancer therapeutic vaccine, recently branded as SaVax. The treatment is based on the combination of a recombinant antigen representative of human vascular endothelial growth factor (VEGF), and clinically tested adjuvants. Pharmacological test in several species effectively demonstrate the therapy effects on inhibiting metastases and tumor growth, inducing specific VEGF- blocking antibodies and specific T-cell responses in several animal species, concomitant with an excellent safety profile. Two sequential phase I clinical trials in solid tumors were authorized by Cuban regulation authorities for conducting studies in humans, to assess safety, tolerance, and immunogenicity, at different antigen doses, and combined with two distinct adjuvants. SaVax was found to be safe and tolerable, with mainly low-grade local adverse effects. Both VEGF specific cellular and humoral neutralizing response were detected in patients receiving SaVax and positivity in more than one test was associated with an increase survival. Patients surviving week 16 in the trials received voluntary off-trial monthly re-immunizations with SaVax, until death, intolerance, marked disease progression, or patient’s withdrawal of consent. No additional onco-specific treatment was administered. After up to 9 years of vaccinations, some individuals experience clinical benefits and 3 of them currently exhibit a complete response: two with originally advance ovarian adenocarcinoma and one with an advance metastatic NSCLC. In the meantime, the compassionate use of the vaccine was authorized in concomitancy with the therapies recommended for the specific malignancies that includes surgery, immunomodulatory agents and chemo- and radio-therapy. Of notice more than 20% of these patients suffer from Ovarian related malignancies (OvCa) and showed some clinical benefits, and a patient suffering from an advance Hepatocellular Carcinoma (HCC) that start on SaVax after tumor relapse, exhibit a complete response. These facts combined with the similarities shared betweenthe mechanism of SaVax induced responses and the antiangiogenics recommended for Ovarian and HCC, encourage us to design and start a phase II/III clinical trial in OvCa. Design of these trials will be presented as well as the current evolution and perspectives for further development of this novel cancer active immunotherapeutic strategy.

39

Where VEGF should be measured? An experience obtained

from phase I clinical trials cancer patients treated with a

VEGF vaccine. Sánchez, J1; Bequet-Romero, M1; Morera, Y1; Hernández, F2; de la Torre, A3; Selman-

Housein, KH4; Martín, Y2; Limonta, M1; Ayala, M1. 1 Department of Pharmaceuticals, Center for Genetic Engineering and

Biotechnology, Havana City, Cuba 2 Department of Clinical Research, Center for Genetic Engineering and

Biotechnology, Havana City, Cuba 3 Department of Clinical Oncology, Celestino Hernández Robau Hospital, Santa Clara,

Cuba 4 Department of Clinical Oncology, Center of Medical and Surgical Research, Havana

City, Cuba javier.sanchez@cigb.edu.cu

CIGB-247 is a VEGF-based vaccine that it has been evaluated in two phase I clinical trials in patients with solid tumors. In both clinical trials VEGF reduction was studied using an indirect methodology named as “Platelet VEGF”. This methodology is based on the estimation of VEGF within platelets by subtracting the plasma VEGF level from the serum level and dividing this by the platelet count, and then this latter expression is additionally corrected by the hematocrit. However, there is broad debate, whether serum or plasma VEGF or other platelet-derived VEGF measurements is the most appropriate strategy to study the changes that occur on ligand bioavailability when patients are submitted to a VEGF-based immunotherapy. The aim of this work is to find out which of these different strategies is the most reliable methodology to investigate the effect of CIGB-247 on ligand bioavailability. We also investigated whether this active immunotherapy is able to reduce VEGF below the levels that can be observed in healthy individuals. “Platelet VEGF” was associated with those groups of individuals that exhibited the best specific humoral response and its variation showed the strongest negative correlation with VEGF-specific IgG antibody levels. For that reason, “Platelet VEGF” was chosen for future investigation of CIGB-247 in phase II clinical trials. The magnitude of platelet VEGF reduction induced by the humoral response elicited by CIGB-247 can be considered as an incomplete blocking effect. Platelet VEGF levels of vaccinated cancer patients can be detected within the physiological range and not below this limit.

40

Antitumoral effects of a VEGF based active immunotherapy

formulated in Alum Phosphate in orthotopic ovarian tumors

in C57Bl/6 mice. I. González-Moya1, C. Canaan-Haden1, S. Rodríguez1, AY Etchegoyen1, D. Pichardo2,

A. Blanco2, J. Sánchez Ramírez1, and M. Bequet-Romero1 1 Department of Pharmaceuticals, Center for Genetic Engineering and

Biotechnology, Havana City, Cuba 2 Animal Facilities, Center for Genetic Engineering and Biotechnology, Havana City,

Cuba isabel.gonzalez@cigb.edu.cu, monica.bequet@cigb.edu.cu

Background: Ascitic fluid is a complication of advanced ovarian cancer (OvCa) in humans that is often related to high VEGF concentration in both serum and ascites itself. Hemorrhagic fluid concomitant to anemic phenotype characterized this disease stage both in humans and in preclinical models. In mice, ID8 model is the choice for testing treatment effects on immunocompetent mice, offering a system that resembles the advanced stages of human OvCa. Data provide herein, aim to characterize the effects of the treatment with a VEGF specific active immunotherapy (CIGB-247) in ascitic fluid accumulation and its association with a specific immune response. Methods: 200 µg of CIGB-247 in Alum phosphate were administered in bi-weekly schedules and animals were challenged with 10 million cells by intraperitoneal injections in both, prophylactic and therapeutic regimes. In order to associate the immunotherapy effects with a particular mechanism, VEGF specific humoral response and neutralization of its binding to VEGFR2 was characterized in serum and ascites using previously described “in house” ELISAs systems. Concentration of leukocytes populations and their activation/senescent markers were studied in ascites by flow cytometry. Growth factors and cytokines related to CD8 mediated immune response were analyzed using ELISA and Multiplex. Results: A significant reduction in animal weight gain, a fact translated to a decrease in ascites load, was observed for administration schemes including at least two doses before the tumor challenge. For this schedule a significant increase in survival was also notice favoring the immunotherapy. Association of ascites and peritoneal tumor loads with the VEGF specific immune response, as well as with the leukocyte phenotype/activation, and cytokine milieu at the tumor compartment will be discussed. Significance: Some surprising complete response and long-term survivals have been reported for CIGB-247 based immunotherapy in patients with advanced OvCa in our phase I clinical trials. We envision our results will shed some light on potential markers enabling the effective translation of this treatment to the clinical setting.

41

Transcriptional evaluation of genes related to the potential

mechanism of action of the VEGF based active immunotherapy

CIGB247, in the context of CT26 ectopic tumors: Preliminary

results. 1Rodríguez S, 2Bakos L., 1 Canaan-Haden C, 1González-Moya I., 1Sánchez-Ramírez J.,

1Morera Y., 2Palenzuela D., and 1Bequet-Romero M. 1 Pharmaceutical Department, 2Pharmacogenomics Group, Center for Genetic

Engineering and Biotechnology, Havana, Cuba. sahily.rodriguez@cigb.edu.cu, monica.bequet@cigb.edu.cu

Background: Multiple mechanisms underlie tumor responsiveness to CIGB-247, an active immunotherapeutic intervention targeting the vascular endothelial growth factor (VEGF). Its effects on humoral and cellular specific response have been characterized in the clinical and preclinical settings. In both cases, most of the work is based on the evaluation of soluble proteins in the systemic compartment. Work presented herein aim to further contribute to characterize CIGB-247 mechanism of action in the tumor compartment. Methods: Quantitative polymerase chain reaction (q-PCR) was used for assessing cytokines, growth factors, immune cells markers and transcriptional factors mRNA expression profile in mice biopsies, obtained from BALB/c mice bearing subcutaneous syngeneic CT26 colon carcinoma treated or not with CIGB-247. CIGB-247 was administered for 6 weeks in a weekly schedule in sNAc-VSSP. After RNA extraction and amplification, transcriptional levels of several genes were analyzed. mRNA levels of IFN-γ, TGFβ1, VEGF-A, CD8 and PD1 were evaluated by qrt-PCR and later confirmed at the protein level using or Flow cytometry. mRNA levels of TGFβ2, VEGF-C, CADH1, CADH2, and Slug proteins known to be involved in epithelial-mesenchymal transition (EMT) were also analyzed using q-PCR. Results: Of interest, mRNA levels were increase for IFN-γ and PD1 in CIGB-247 treated tumors in agreement with ELISA results and Flow cytometry data. Nevertheless, further characterization of PD1 MFI in both CD4 and CD8 tumor infiltrating cells indicates that the vaccine treatment induces a population of T cells with a reduce PD1 density at the membrane pointing to an early activation state for these cells within the tumor microenvironment. Some disparities where however observed for CD8 expression that show no difference between groups even when by flow cytometry a significant increase in this cell type was observed in tumors from CIGB-247 treated animals as compare to placebos. Contrary to some reports on antiangiogenics VEGF-A, VEGF-C and TGFβ1 mRNA expression was not altered by the vaccine administration. Protein levels of VEGF and TGFβ1 were however significantly reduced, pointing to a favorable antiangiogenic microenvironment in CIGB-247 treated animals. Antiangiogenic treatments have been related to a pro-metastatic phenotype, but in the case of CIGB-247 the expression of N-Cadherin or the transcription factors governing transition to an EMT phenotype snail and slug were not affected. E-Cadherin mRNA expression was in turn significantly induced in the tumors treated with CIGB-247, indicating the potential predominance of an epithelial phenotype within the tumor. Conclusions: These results offer novel insights into the differentiated mechanisms of CIGB-247 induced responses, as compare to other antiangiogenics; pointing to the need for confirm some mRNA variations in terms of proteins and expand these evaluations to a higher number of genes and proteins and other tumor models.

42

ELISA method for quantification of antibodies against VEGF

protein. Coizeau, E1; Avila, L1; Chávez, S1; Freyre, G1; Vázquez, A1; Sánchez, J2; Bequet-

Romero, M2 and Lemos, G1. 1Department of Chemistry-Physics. 2Pharmaceutics Department. Center for Genetic

Engineering and Biotechnology, Havana, Cuba. edelgis.coizeau@cigb.edu.cu

Tumor angiogenesis is a hallmark of cancer, a key event that governs tumor

progression and metastasis. A major driver of the angiogenic process is the vascular

endothelial growth factor (VEGF) which is often found overexpressed in tumors.

HEBERSaVax is a cancer therapeutic vaccine candidate based in the use of

representative fragments of VEGF, a novel therapeutic approach to induce antibodies

that neutralize the ability of VEGF to interact with its specific receptors. In this work

we present the establishment of a quantitative test based in a previously developed

semi-quantitative method. Briefly, a Reference Material composed of a mixture of

high titer anti-VEGF human sera was quantified and certified. Antibodies levels were

expressed in arbitrary units per milliliters. Calibration curve was analyzed using four

parameters non-logarithmic regression (4PL). The calibrator linearity range was

obtained at antibodies titer from 0.38 to 100 AU/mL. In validation experiments the

test fit for purpose, showing high accuracy and precision, being currently applied as

an analytical tool in quantification of anti-VEGF antibodies levels in the clinical trial

samples.

43

Clinical Research of CIGB-300 in patients with Locally

Advanced Cervical Cancer

Idania Baladrón, Ana Margarita Solares, Miguel Sarduy, Gustavo Crespo, Luis Alfredo Estévez, Doris González Díaz, Agueda Santana, Jorge Luis Soriano, Noyde

Batista, Adlin Lopez, Yasser Perera, Hernán Farina, Carmen Valenzuela, Pedro López Saura, Ignacio Hernández, Idrián García, Lidia Gonzalez, Aley Palau, Ignacio

Hernández, Idrián García, Alina Díaz, Carlos López, Silvio E Perea, Verena Muzio. Center for Genetic Engineering and Biotechnology, Havana 10600, Cuba. “Hermanos

Ameijeiras Hospital, Havana, Cuba. National Center for Toxicology, Havana, Cuba. Clodomira Acosta Gynecobstetric Hospital, Havana, Cuba.

CIGB-300 is a novel synthetic proapoptotic peptide that inhibits CK2-mediated

phosphorylation. It affects cell viability and proliferation, as well as angiogenesis,

metastasis, and synergizes with more conventional anticancer drugs including

paclitaxel, cisplatin, and with a targeted therapy such as erlotinib. The initial clinical

development of CIGB-300 has been focused on cancer of the uterine cervix that

allowed for intratumor injections. Phase 1 clinical trials in women with HSILs and

locally advanced cervical cancer established the MTD and DLT with and without

antihistamine pre- medication and verified high drug tumor accumulation with intra-

tumor injections. Optimization of CIGB-300 intratumor injections suggest that

application of 0.5-mL injections equidistant from the tumor center result in the

highest tumor accumulation of CIBG- 300. Furthermore, downregulation of

B23/nucleophosmin in cervical tumors following the administration of CIGB-300 can

be considered as a potential biomarker of response in future clinical trials. Although

not mandatory for CIGB-300 doses ≤70 mg, premedication with antihistamines

improves tolerability and would be recommended in future clinical trials assessing

efficacy. Complete response rate data suggest the response rate achieved

administering CIGB-300 with chemoradiotherapy is higher than that attained with

single-agent CIGB-300 in the neoadjuvant setting, such that combination therapy

should be further explored in efficacy studies. The CIGB-300 clinical research data

summarized here can be used to support phase 3 clinical trials of this novel agent in

locally advanced cervical cancer.

44

Effect of the clinical-grade Casein Kinase 2 inhibitor CIGB-300 on

NF-kB signaling in Acute Myeloid Leukemia cells Ramon AC1*, Vázquez-Blomquist D2, Besada V3, Rosales M1,4, Wisniewski J5,

González L. J3, Perera Y1, Perea SE1 1 Molecular Oncology Group, Pharmaceuticals Department, Center for Genetic

Engineering and Biotechnology. Havana, Cuba.2 Pharmacogenomic Group, System Biology Department, Center for Genetic Engineering and Biotechnology. Havana,

Cuba.3 Proteomic Group, System Biology Department, Center for Genetic Engineering and Biotechnology. Havana, Cuba.4 Animal and Human Biology

Department, Faculty of Biology, University of Havana. Havana, Cuba.5 Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry.

Munich, Germany. ailyn.ramon@cigb.edu.cu

CIGB-300 is a proapoptotic peptide that impairs the Casein Kinase-2-mediated phosphorylation. The major target of CIGB-300 in solid tumors is the nucleolar protein B23/nucleophosmin, however in Acute Myeloid Leukemia (AML) the action of CIGB-300 could be mediated by alternative mechanisms, involving other molecular targets and signaling pathways. Global analysis of gene expression using microarray technology evidenced a possible effect of CIGB-300 on the NF-kB signaling pathway in AML cell lines. Therefore, it is important to validate this finding in order to elucidate the CIGB-300 mechanism in AML. We performed qPCR assay to validate NF-kB target genes modulated by CIGB-300 according to microarray data in AML cell lines. Western blot and ELISA experiments were conducted to evaluate CIGB-300 effects on NF-kB downstream proteins and TNF-α induction in AML cells lines, respectively. We demonstrated that CIGB-300 increases some NF-kB targets like TNF-α, IkB and p50 at the mRNA level indicating a possible activation of NF-kB signaling pathway. Additionally, it was evidenced an effect of CIGB-300 on activation of NF-kB pathway which could be explained by a decrease of the NFKB inhibitor at protein level. CIGB-300 also increases TNF-α secretion, an inducer and target protein of this signaling pathway. NF-kB is one of the signaling pathways modulated by CIGB-300 in AML. Altogether, our data provides new insights into CIGB-300 mechanism of action in AML.

45

NPM1c+ as a putative novel target of interaction for the

antitumoral peptide CIGB-300

Pérez GV1*, Ramon AC1, Rosales M1,2, Perea SE1 and Perera Y1 1 Molecular Oncology Group, Pharmaceuticals Department, Center for Genetic

Engineering and Biotechnology. 31 Avenue / 158 and 190, Cubanacan, Havana 10 600, Cuba.

2 Animal and Human Biology Department, Faculty of Biology, University of Havana. 25 street No.455, Vedado, Havana 10 400, Cuba.

george.perez@cigb.edu.cu

Acute myeloid leukemia (AML) is the most common leukemia in adults and among the most lethal worldwide. Many protein kinases are established targets in cancer therapy and several kinase inhibitors are already undergoing clinical trials. CIGB-300 is a synthetic peptide that impairs phosphorylation by casein kinase 2 (CK2) through targeting the substrate’s phosphoacceptor domain. One of these substrates is the chaperone nucleophosmin 1 (NPM1) which plays an important role in the maturation of ribosomal RNA, reason why it can shuttles between the cytosolic and nucleolar compartments. Preliminary evidences suggested that serine 125 residue on the NPM1 protein is targeted by CIGB-300 in various tumor cell lines, which was also supported by co-localization of this peptide with NPM1 at the nucleolar compartment. The mutated variant NPM1c+ is the most common mutational event in AML. Of note, a distinctive property of NPM1c+ is its aberrant cytosolic location where it triggers its oncogenic potential. In this work, the NPM1c+ variant was transfected in HEK-293 cells and stable expression model was generated by selection with antibiotic G418. The expression of NPM1c+ in this cellular context was confirmed by qPCR and Western blot. In order to demonstrate the possible interaction between the NPM1c+ protein and the CIGB-300 peptide, pull-down assays were carried out. Importantly, our data provide the first experimental evidence demonstrating that CIGB-300 interacts with NPM1c+ and also, it reinforces the molecular basis that could support the feasibility of CIGB-300 in treating AML patients with this mutation.

46

MODULATION OF PD-L1 EXPRESSION BY CIGB-300 IN HUMAN

CERVICAL CANCER CELL LINES

Aguilar-Noriega D1, Hurtado D1, Vazquez D1, Perea SE1. Affiliation: 1Center for Genetic Engineering and Biotechnology, Ave 31, 158 and 190,

Playa, 10600, P.O. Box 6162, Havana, Cuba. daylen.aguilar@cigb.edu.cu

Cancer cells are sometimes able to evade the host immunity in the tumor microenvironment. One of the principal mechanisms is constituted by immune checkpoint. One of the most critical pathway in this system is the tumor immune suppression mediated by the programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1). PD-L1 is expressed in a wide variety of tumors and interaction with PD1, on immune cells, produce anergy and apoptosis of lymphocytes, allowing tumor development. In this work we evaluated PD-L1 levels modulation by the CK2 inhibitor, CIGB-300 peptide in cervical cancer cell lines. The cervical cancer cell lines Caski and SiHa were treated with 80µM and 130µM of peptide, respectively, during 5, 8 and 24 hours. PD-L1 mRNA levels was quantified by qPCR assay and protein expression was evaluated by flow cytometry. IFN-γ was employed as positive control for PDL-1 induction. Data showed the CIGB-300 induced significant increase of PD-L1 mRNA levels in Caski cell line although no significant increase was observed in protein levels. Otherwise, the undetectable baseline levels of PD-L1 mRNA in SiHa cells were mot modified by CIGB-300. In conclusion, these preliminary results indicate that CIGB-300 can modulate the PD-L1 mRNA levels in tumor cells with detectable but not with undetectable baseline levels of this immunocheckpoint. Additional time-course experiments monitoring PDL-1 protein levels are needed to correlate it with the mRNA upregulation induced by CIGB-300 in CaSki cells.

47

MultiOMICs data analysis enables identification of biological

processes and pathways modulated by CIGB-300 in leukemia cells Rosales M1,2 *, Vázquez-Blomquist D3, Besada V4, Ramón AC2, Perera Y2, Perea SE2

1 Department of Animal and Human Biology, Faculty of Biology, University of Havana. Havana, Cuba.

2 Molecular Oncology Group, Department of Pharmaceuticals, Center for Genetic Engineering and Biotechnology. Havana, Cuba.

3 Pharmacogenomic Group, Department of System Biology, Center for Genetic Engineering and Biotechnology. Havana, Cuba.

4 Proteomic Group, Department of System Biology, Center for Genetic Engineering and Biotechnology. Havana, Cuba.

mauro.rosales@fbio.uh.cu CIGB-300 is an anticancer peptide conceived to block Casein Kinase 2 (CK2) mediated

phosphorylation, molecular event that has emerged as an attractive therapeutic

target in hematological malignancies. Since CK2 phosphorylates a large number of

substrates, we attempted to identify biological processes and pathways regulated by

CIGB-300 in Acute Myeloid Leukemia (AML) cells, as well as potential targets for the

peptide. In this work, we performed a global analysis of genes and proteins

modulated by CIGB-300 in two AML cell lines using microarray gene expression,

proteomic and phosphoproteomic approaches. Biological processes, pathways and

protein kinases related to modulated genes, proteins and phosphopeptides were

identified by enrichment analysis using DAVID 6.8, Toppfun and KEA2 web-based

tools. Gene expression and protein level analysis showed that CIGB-300 modulates

genes and proteins involved in cell death and cell cycle, but also in myeloid

differentiation and inflammatory response. Those biological effects could be

explained by the impact of the peptide in TNF, NF-kB and MAP Kinase pathways.

Additionally, phosphoproteomic analysis corroborated the biological processes and

pathways previously identified, and provided a list of CK2 substrates as potential

targets for this CK2 inhibitor. Further studies to validate the achieved results need to

be conducted in order to completely elucidate the mechanism of action of CIGB-300

in AML cells. Altogether, our findings indicate the biological processes and pathways

regulated by CIGB-300 in AML cells, provide potential targets for this peptide, and

also reinforce the potentialities and rationality of CK2 pharmacologic inhibition for

AML targeted therapy.

48

Phosphoproteomic profile modulated by the anticancer peptide

CIGB-300 in human promyelocytic leukemia (HL-60) cells.

Rodríguez-Ulloa, A.(1)*; Perera, Y.(1); Ramos-Gómez, Y.(1); González, L.J.(1); Wisniewski, J.(2); Perea, S.E.(1); Besada, V.(1)

1 Proteomics Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba

2 Department Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Germany

arielis.rodriguez@cigb.edu.cu

CIGB-300 is a proapoptotic peptide-based drug that abrogates the CK2-mediated phosphorylation. This peptide has antineoplastic effect on chronic lymphocytic leukemia cells in vitro and in vivo. To understand the mechanisms involved on such anticancer activity, the HL-60 cell line phosphoproteomic profile modulated by CIGB-300 was investigated using a label-free phosphoproteomics approach. Functional enrichment, biological pathways and network of enzyme-substrate relationships were analyzed using bioinformatics tools. Our analysis finds 3900 unique phosphosites on 1961 proteins. The majority of the identified phosphosites were represented by phosphoserine (87.2%). A total of 140 phosphosites (3.6% of the dataset) on 120 phosphoproteins were significantly regulated (p-value ≤ 0.05, Fold-change (CIGB-300/Control) cut-off ≥ 1.5) in CIGB-300-treated cells. Phosphoproteins related to transcription, RNA processing, cell cycle, cell death, translation and ribosome biogenesis were significantly modulated in response to CIGB-300 treatment. The nuclear proteins (77% of the dataset) were the most represented in the phosphoproteome. CIGB-300 treatment decreases the phosphorylation level of 18 CK2 substrates, including Nucleophosmin and Nucleolin which have been previously validated as potential CIGB-300 targets. Additionally, PI3K/AKT-, mTOR- and MAPK- signaling pathways seem to be associated with a decreased phosphorylation of target proteins in CIGB-300-treated cells. Phosphoproteomics analysis provides evidences supporting the CIGB-300 molecular mechanism of action in leukemia cells.

49

The influence of different peptide to increase the

immunogenicity of the GnRH vaccine for prostate cancer

treatment.

Fuentes F, Junco J, Calzada L, Bover E, Serradelo JA, Hernández E, Pimentel E, Basulto R, Reyes O, Garay H and Guillen G

Center for Genetic Engineering and Biotechnology, Camagüey. Cuba Center for Genetic Engineering and Biotechnology, Habana. Cuba

franklin.fuentes@cigb.edu.cu

Therapeutic vaccines, specifically the Gonadotrophin Releasing Hormone (GnRH) vaccine, are considered an additional therapeutic option for advanced stage prostate cancer. Our work showed amplification of the immune response when combining two peptides with and without the Very Small Size Proteoliposomes (VSSP). VSSP is a potent adjuvant for dendritic cells activation and Th1 differentiation as enhanced immune response. Methods: The test was carried out in Copenhagen rats as animal model. Resultst: The use of both peptides and their combination with VSSP generated a potentiation of the immune response statistically superior, in term of generating anti GnRH antibody and effects on target organs, when it was compared with the effects which occurs with independent peptides and with and without the VSSP. These results can find application in the development of GnRH vaccine candidates and in peptide based vaccine strategies. Conclusions: Immunization with the peptide combination enhances the immune response when mixed with the VSSPs

50

Bioequivalence of natural and synthetic VSSP using the

peptide GnRHm1/TT for the treatment of prostate cancer. Calzada L, Fuentes F, Junco J, Campal A, Garay H, Serradelo A, Lopez E and Guillén G

Center for Genetic Engineering and Biotechnology, Camagüey. Cuba Center for Genetic Engineering and Biotechnology, Habana. Cuba

lesvia.calzada@cigb.edu.cu

Therapeutic vaccines, specifically the Gonadotrophin Releasing Hormone (GnRH) vaccine, are considered as an additional therapeutic option for the treatment of prostate cancer in advanced stage. Our work showed the Bioequivalence of natural and synthetic Very Small Size Proteoliposomes (VSSP) used as enhancers of the immune response in the Heberprovac, vaccine candidate for the treatment of prostate cancer. The use of both VSSP showed no significant differences in the generation of anti-GnRH antibodies and on the effects on the prostate and testes as target organs of the treatment. This study was carried out in male Copenhagen rats which were immunized whit 750 Ug of the GnRHm1/TT with natural and synthetic VSSP. Synthetic VSSP can be considered bioequivalent in enhancing the immune response of the peptide GnRHm1-TT for prostate cancer in terms of immungenicity and effect on target organs.

51

Establishment of an ELISA for the quantification of the CIGB-

552 peptide in plasma samples from patients of ARGOS Phase

I Clinical Trial.

Vázquez, A1; Sobrino, X1; Cabrales, A1; Támbara, Y1; Guerra, M2 and Lemos, G1. 1Department of Chemistry-Physics. 2Pharmaceutic Department. Center for Genetic

Engineering and Biotechnology, Havana, Cuba. amalia.vazquez@cigb.edu.cu

This work describes the establishment of a test for the quantification of the CIGB-552 peptide in plasma samples from patients of the ARGOS Phase I clinical trial. With this objective a sandwich ELISA was established, where a polyclonal antibody obtained in rabbit was used in the capture, and a mAb in the detection, both generated against the CIGB-552 peptide. The CIGB-552 peptide was used as reference material for the assay, and the results were processed making a linear regression with the statistical package GraphPad v6.1 for the calculation of the peptide concentrations in the samples. The linear range of the curve was obtained from 50 to 0.05 ng/mL and the lower quantification limit estimated was 3.13 ng/mL. This assay allowed quantifying the presence of the CIGB-552 peptide in samples of immunized patients. Results obtained were used in the pharmacokinetics studies.

52

Studies of different doses of the CIGB550-E7 vaccine

candidate with a fixed dose of Al(OH)3 in the TC-1 model.

Rodríguez N1, Alfonso AB1, Granadillo M1, Batte A1, Torrens I1 1 Pharmaceutical Department. Center for Genetic Engineering and Biotechnology,

Havana, Cuba. nadia.rodriguez@cigb.edu.cu

Cervical cancer is the second leading cause of death from cancer in women between the ages of 15 and 44. The development of this disease is generally associated with persistent infection with the high-risk viral types of human papillomavirus (HPV). In particular, HPV16 contributes to 50% of cervical cancer cases reported annually. The therapeutic vaccine candidate (CIGB550-E7) was designed for treatment of pre-cancerous lesions and cancer associated with persistent infection by HPV16. This candidate comprises the fusion between a mutein of HPV16 E7 protein with a cell-penetrating peptide (CIGB550) with immunostimulatory properties. In previous studies in the TC-1 model we have observed that the combination of the CIGB550-E7 with VSSP (Very Small Size Proteoliposomes), a Th1 polarizing adjuvant, generates a potent antitumor response thus increases the survival of the mice. Also, in preclinical results using adjuvants based on aluminum salts, specifically Al(OH)3 (alum), we obtained similar results to those obtained with VSSP. In this work a comparative study of different doses of the CIGB550-E7 adsorbed in fixed dose of Al(OH)3 was carried out to determine the best combination as alternative to be used in clinical trials with this candidate. The four doses used (60, 120, 240 and 480 µg), showed no statistically significant differences, so for future human study was select as the best combination 120 µg of this candidate with 0.6 mg of Al(OH)3.

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Establishment of T-lymphocytes quantification with triple-

labeled antibodies by flow cytometry in samples from

patients of ARGOS phase I clinical trial.

Avila L1, Vázquez A1, Freyre F1, Martinez R1, Guerra M2 and Lemos, G1. 1 Biomedical Research, Center for Genetic Engineering and Biotechnology (CIGB)

Avenue 31, P.O. Box 6162, Havana 6, 10 600, Cuba lismary.avila@cigb.ecu.cu

T lymphocytes play a fundamental role in the evaluation of different pathologies that allow the diagnosis, prognosis and clarification of the physiopathology of certain diseases such as cancer, which are characterized by alterations in the number of lymphocytes subsets. In this work we report the implementation of a methodology for the analysis and quantification of cell subpopulations in whole blood of clinical trials patients and the gating strategy of analysis by flow cytometry, using a commercially available cocktail of three monoclonal antibodies, anti-human CD4, CD8 and CD3, conjugated with the fluorophores FITC, PE and PE-Dy647 respectively. This methodology allowed quantification by applying in real time the absolute count in small volumes of human whole blood. The developed method is fast, feasible, requires little manipulation and a small volume of the patient's whole blood. The method allowed monitoring T-cells population in treated patients.

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Farmacometric study of nimotuzumab in colorectal cancer Fernández B1, Rodríguez L2, de Castro N2, Lavastida A4

1. Production Department, Center for Genetic Engineering and Biotechnology, CIGB. Ave 31 be/ 158 and 190, Cubanacán, Playa, POBox

6162, Havana, 10600, Cuba

2. Department of Pharmacology. Institute of Pharmacy and Food, University of Havana. Ave. 222 and 23, La Lisa, Havana, 10600, Cuba

3. Department of Clinical Trials, Center of Molecular Immunology. Street 216 and 15 Ave. Atabey, Playa, Havana, Cuba

betsy.fernandez@cigb.edu.cu

Colorectal cancer is one of the most frequent human cancers worldwide. More than 30 years ago, the development of biotechnology has facilitated the acquisition of new products for the treatment of cancer, an example of which is the generation of monoclonal antibodies (mAbs). The nimotuzumab is a humanized monoclonal antibody targeted to the extracellular domain of human epidermal growth factor receptor. The purpose of this work is to develop a population pharmacokinetic (PopPK) model for nimotuzumab in patients with colorectal cancer. A Phase I and non-controlled open clinical study was carried out in patients with colorectal cancer. In this study, 19 patients were divided into four groups at each of the following fixed dose levels: 200, 400, 800, 1200 mg. Blood samples were drawn during 11 days for PK assessments. PopPK analysis of 197 concentration-time data from 19 patients was performed using the nonlinear mixed-effect model approach with NONMEM 7.3. The pharmacometry was evaluated by using the Quasi-steady state approximation (QSS) of the complete receptor-mediated drug disposal (TMDD). Interindividual variability was modeled assuming a lognormal distribution, and was associated with central volume of distribution (Vc). The volume of population distribution was 4,769L / h with an interindividual variability of 63%. The developed PopPK model can be used to guide the dose selection for nimotuzumab during routine clinical practice in patients with colorectal cancer. The model will further support the ongoing investigations of the PK/PD relationships of nimotuzumab to improve its therapeutic use.

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Pharmacophore modelling of PD-L1 immune checkpoint protein

through molecular dynamics simulations

Montes, L.1; Mejías, C.1; Guirola, O.1 1 Bioinformatic Group, System Biology Department, Center for Genetic Engineering

and Biotechnology, Havana, Cuba. laura.montes@cigb.edu.cu

Due to the clinical success of cancer immunotherapy, the design of PD-1/PD-L1 inhibitors has become an area of active research. To date, only five monoclonal antibodies are approved by FDA. Despite the great effort for the development of small molecules and peptides as inhibitors, only one has reached clinical trials. Pharmacophoric models are a proven useful tool for drug design. The effectiveness of receptor-based pharmacophore modeling is limited due to the neglect of protein flexibility and desolvation effects. In this study, we performed co-solvent molecular dynamics simulations of PD-L1 protein in order to obtain a pharmacophore model of PD-L1 immunecheckpoint protein. Analyzing the affinity of probe molecules by PD-L1, we obtained the interface of PD-1/PD-L1 as a zone with the highest convergence of probe molecules. The interactions established with PD-L1 residues varied from hydrophobic interactions to hydrogen bonds and salt bridges with critical residues for PD-1/PD-L1 binding (M115, I116, D122, Y123, I54, Y56, E58, Q66). The superposition of known inhibitors of PD-L1 as Peptide-57, BMS-1166 and KN035 nanobody allows us to validate the pharmacophore model due to the excellent correlation with its features. The use of this pharmacophore can help to enhance binding affinity of molecules for PD-L1.

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Cervico-uterine cancer detection and prevention: next steps in the

era of precision medicine.

González, N; Díaz, M; García, G; Ortega, A. Molecular Genetic Department, Hermanos Ameijeiras Hospital, La Habana, Cuba.

nadezhdagonzalez@gmail.com

Human papillomavirus (HPV) has been identified as a major factor that leads to cervical cancer. Their prevention and early detection innovations have begun moving towards the integration of molecular knowledge and risk stratification profiles to allow for a more accurate representation of at-risk individual, known at present as precision medicine. The first approximation in this sense were done in “Hermanos Ameijeiras” Hospital with the introduction of cobas® 4800 Human Papillomavirus (HPV) Test (Roche) for diagnosis of HPV. A screening study was conducted in order to identify the possible presence of papilloma virus in women with previous negative cytology. The test utilizes amplification of target DNA by the Polymerase Chain Reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk (HR) HPV types in a single analysis. The test specifically identifies HPV16 and HPV18 and the other high risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). The specimens to cervical cells collected in cobas® PCR Cell Collection Media (Roche). A total of 929 asymptomatic women, between 30-50 years, with previous negative cytology, were screened. A 16.1% (150 women) positive for HPV was obtained. 13.3% (20 women) were positive for genotype 16, 10.7% (16 women) positive for genotype 18. 76% were positive for the other high-risk genotypes. This work demonstrates that screening of HPV should be performed in Cuba as precision medicine for early detection and prevention of cervical-uterine cancer.

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EGFR mutations as biomarker to target therapies in patients with

lung cancer in Cuba.

Ortega A, Garcia G, Hernández B Department of Molecular Genetics, Hermanos Ameijeiras Hospital, Havana City,

Cuba. alexanderortega81@gmail.com

Cancer is one of the leading causes of death in Cuba. Around 40 thousand new cases are diagnosed each year in the country, more than 2000 are from lung cancer. The new generation treatments, designed to inhibit the growth, progression and spread of the tumor, are based on the modification of the white proteins associated with them. In this sense, mutations in EGFR have been identified among more than 380 prognostic genes and predictors of treatment response. Our goal was to identify target mutations in the EGF receptor susceptible to treatment with TKI. Samples of lung adenocarcinoma biopsies embedded in paraffin were used to extract the DNA using the commercial DNA preparation KIT and liquid biopsy samples were processed from cfDNA preparation KIT; PCR was performed in the COBAS 4800 system (Roche) with the use of commercial IVD and CE kits to detect EGFR mutations. Negative and positive controls were valid for all PCR runs. The positivity for EGFR in Cuba is still uncertain, since several preanalytical variables are involved, we have patients susceptible to TKI and resistance to it is monitored by monitoring the liquid biopsy. In Cuba, a novel method was implemented for the determination of genetic biomarkers by means of real-time PCR, which opens the way to precision medicine in cancer.

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HLA Polymorphism in Havana’s population. Chang, A1; Marcell, L; Rodríguez E; Figueras JP; Guevara, LM; Valdés, R; Ustariz, CR.

Histocompatibility department, Institute of Hematology and Immunology, La Habana, Cuba.

achangm@gmail.com

A retrospective study was carried out in the Histocompatibility department of the Institute of Hematology and Immunology (IHI), with the aim of characterizing the polymorphism of the human leukocyte antigen (HLA) system in the Havana population. A non-probabilistic sample was used that included the HLA of 222 cadaveric donors from all hospitals in Havana, from January 2013 to January 2019. From a blood sample, 75 HLA genes had been previously typed in each deceased using low resolution Polymerase Chain Reaction – Sequence Specific Primers (PCR-SSP). The Arlequin 3.5.2.2 program was used for the immunogenetic analysis. The most frequent genes were A*02, A*24, B*07, B*44, DRB1*13, DRB1*15, DQB1*02 and DQB1*03 and the most frequent haplotypes were HLA-A*29 B*44 DRB1*07 DQB1*02, A*02 B*44 DRB1*15 DQB1*06 and A*02 B*35 DRB1*04 DQB1*03. The polymorphism in the genes of the HLA system in the citizens of Havana presents characteristics similar to those described in the Cuban population, and in the populations of the rest of Latin America and the world, although with distinctive characteristics at the level of haplotypes.

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Improved patent portfolio associated to the CK2-inhibitor peptide

CIGB-300 fostered by bioinformatics and OMICS Gonzalez, Sa; Perea, Sb; Perera, Yb; Rodriguez, Ac; Besada, Vc; Gonzalez, LJc;

Rodriguez, Rc; Mazola, Yc; Musacchio, Ac; Vazquez, Ma; Ubieta, Ra. a Patent Department, b Department of Pharmaceuticals, c System Biology

Department, Center for Genetic Engineering and Biotechnology (CIGB), La Habana, Cuba.

sonia.gonzalez@cigb.edu.cu

Protein kinase 2 (CK2) is a serine-threonine kinase that has emerged as a potential anticancer target. Researchers at CIGB, Cuba, firstly identified 11 peptides able of impairing the phosphorylation by CK2. It gave place to a patent application disclosing such peptides and their use as pharmaceuticals. It was demonstrated that a chimera named CIGB-300, composed by one of those cyclic peptides and the Tat cell-penetrating peptide, was able to induce tumor regression when injected directly into solid tumors in mice. Encouraged by said results, in silico molecular modelling was employed, and dozens of chemical compounds were identified based on their ability to block the phosphoaceptor site on the CK2 substrates. This new invention was reflected in a second application owned by CIGB, disclosing the use of said compounds in the preparation of drugs for cancer treatment. In addition, a proteomic study was conducted with CIGB-300, finding that this peptide unexpectedly regulates a group of proteins on tumor cells. The use of said approach allowed explaining the synergistic antitumor effect of the peptide with cytotoxic drugs, comprised in a third invention having CIGB as an applicant. All three inventions were filed internationally, in three patent families with more than 100 patent documents. Hence, in silico molecular modelling and proteomics allowed to foster the patent portfolio around CIGB-300, a peptide that exhibits anticancer properties in vitro and in vivo.

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CIGB patent portfolio for cancer project research Vazquez M.1, Gonzalez S.1, Ubieta R.1, Perea S.2, Rodriguez R.2, Bequet-Romero M.2, Abrahantes M.C3, Bello I.4, Guerra M.2, Torrens I.2, Junco J.2, Chinea G.2, Lamdan H.2

1 - Patent Department; 2 – Biomedical Research Department; 3- Technological Development Department, 4- Clinical Research Department, Center for Genetic

Engineering and Biotechnology, Havana, Cuba. mariela.vazquez@cigb.edu.cu

In 2018, 18.6 million new cases of cancer were recorded worldwide, a little more than half fatal. In America, it represents the second cause of death and, by 2030, PAHO expects this condition to increase by 32 percent, mainly due to population aging and exposure to risk factors. Cuba is one of the nations with one of the highest incidence rates of this disease in the world, which constitutes the second cause of death. According to the NATIONAL CANCER REGISTRATION of the Ministry of Public Health (2018), in the last 15 years the risk of getting sick and dying of cancer in Cuba has increased in the age groups between 35 and 64 years. For this reason, investment and research in Cuba is encouraged in this area. In line, at the biomedical research area of the Center for Genetic Engineering and Biotechnology, a wide range of projects dedicated to the Oncology branch are developed, whose innovative results have been subjected to patent protection. The present work shows the CIGB patent portfolio related to the research and development of therapeutic products for cancer. The CIGB patent portfolio associated with cancer is composed by 12 patent families granted and in force in more than 40 countries in the world. All together the CIGB cancer patent portfolio comprises more than 300 patent documents.

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