Keren Paz

Cyclin D1 Is Transcriptionally Regulated by and Required for Transformation by Activated Signal Transducer and Activator of Transcription 3

Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer-derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1(-/-) and cyclin D1(-/+) fibroblasts led to anchorage-independent growth of only cyclin D1(-/+) cells. Furthermore, knockdown of cyclin D1 in breast carcinoma cells led to a reduction in anchorage-independent growth. Phosphorylation of the retinoblastoma (Rb) protein [a target of the cyclin D1/cyclin-dependent kinase 4/6 (cdk4/6) holoenzyme] was delayed in the cyclin D1(-/-) cells relative to cyclin D1(-/+) cells. The E7 oncogene, whose activity includes degradation of Rb and dissociation of Rb from E2F, did not confer anchorage-independent growth to the cyclin D1(-/-) cells but, in conjunction with vSrc, resulted in robust growth in soft agar. These results suggest both a cdk-dependent and cdk-independent role for cyclin D1 in modulating transformation by different oncogenes.

The TGFβ–miR200–MIG6 Pathway Orchestrates the EMT-Associated Kinase Switch That Induces Resistance to EGFR Inhibitors

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor β (TGFβ)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFβ-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFβ-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

Recent advances in targeted therapy of human myelogenous leukaemia.

Despite major advances in the diagnosis and treatment of myelogenous leukaemia during the past few decades, this group of diseases remains a serious medical concern with > 15,000 new cases each year and a mortality rate of ∼ 10,000 in the US alone. Current available conventional therapies, including chemotherapy and bone marrow transplantation, often cause severe side effects owing mainly to the lack of specificity of the treatment. In the past years, significant progress has been made towards understanding the pathogenesis of myelogenous leukaemia from the molecular standpoint. To this end, a growing number of approaches are being exploited for the identification and validation of new therapeutic targets suitable for more potent and specific or ‘targeted’ intervention. In this review, the authors focus their discussion on the four most promising myelogenous leukaemia-associated molecular targets currently being pursued by major pharmaceutical and biotechnology companies, fms-like tyrosine kinase 3 (FLT3), CD33, farnesyl transferase and BCR-Abl, with emphasis on recent progress on the clinical development of therapeutic agents, including both kinase inhibitors and monoclonal antibodies, to these targets.

Abstract 1190: Multifactorial biological processes govern engraftment of patient-derived tumor tissue in immunodeficient mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA TumorGrafts (also known as patient-derived xenografts) are a valuable tool for the personalization of oncology treatment, as well as development of new cancer therapeutics. Tumor explants are engrafted into immunodeficient mice and allowed to develop prior to screening against a panel of drugs or drug combinations to assess which best inhibit tumor growth. These models capture the chaotic heterogeneity, histopathology, and biology of the original tumor, as well its 3-dimensional interaction with the surrounding stroma and other cells migrating into the tumor environment. TumorGrafts will serve basic and clinical research groups as an increasingly valuable preclinical model of cancer. One important variable governing the generation of these models is the take rate, or the percentage of patient tumors that successfully engraft and grow in the mice. This is a potentially critical limitation to applying these preclinical models for improving patient treatment and advancing novel drug regimens to the clinic. Hence, there is a need to understand and exploit the mechanisms that influence take rate in order to ensure that the majority of tumor explants readily engraft and expand. We describe here our experience in optimizing the engraftment of patient tumor tissue in immunodeficient mice. We found a number of factors contribute to take rate including tumor type, tissue quantity and quality, engraftment site, oxygenation state, neovascularization and the presence of extracellular stromal components and cells. We have also uncovered a correlation between the growth rate of tumors in the mice and the clinical aggressiveness of the original malignancy, information that may be useful in guiding clinical management. Moreover, we describe how we are now able to consistently use biopsy material rather than surgical explants to establish TumorGrafts, a crucial step forward that allows this technology to benefit patients diagnosed with early-stage cancers or where surgery is not indicated. Although engraftment of patient tumor tissue in immunodeficient mice is a complex process, with multiple factors impacting success, we have effectively optimized this process, improving our take rate and at the same time, reducing the time to obtaining drug screening results, all without compromising tumor integrity. Citation Format: David M. Vasquez-Dunddel, Gilson Baia, Amanda Katz, Daniel Ciznadija, David Sidransky, Keren Paz. Multifactorial biological processes govern engraftment of patient-derived tumor tissue in immunodeficient mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2014-1190

Abstract 2793: Development of an androgen-dependent prostate Champions TumorGraft™ cancer model.

Prostate cancer (PCa)is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males, with the highest incidence rates recorded in developed countries such as those in North America (Jemal et al., 2011). While the five year-survival rate for PCa patients with localized disease is 100%, it is only 30.6% once the cancer metastasizes (Vishnu et al., 2010). One of the biggest challenges for finding a better treatment for metastatic PCa is the lack of predictable and accurate preclinical models that closely recapitulate different stages of the pathogenesis of human PCa. Champions Oncology focuses on the development of Champions TumorGraft™ models derived from the direct implantation of patient tumors into immunocompromised mice. Compared to traditional cell line-based xenograft models, the patient-derived TumorGraft models maintain stable gene-expression patterns and mutational status and correlate to clinical predictability. To overcome the challenge for PCa, Champions Oncology has utilized its TumorGraft technology to establish and characterize a new androgen-dependent prostate cancer TumorGraft model, CTG-0488. This involved the implantation of a primary human prostate tumor in immunocompromised mice in a manner that preserves the biological properties of the original human tumor. In this preliminary work, the characteristics of Champion TumorGraft™ model CTG-0488 are showcased, including tumor histology, mutational status, and chemosensitivity profiles to several standard of care compounds. Overall the development of an androgen-dependent prostate cancer Champions TumorGraft™ model will significantly aid in the development of oncology compounds focused on androgen-dependent prostate cancer. Citation Format: Tin Oo Khor, Keren Paz, Dhanrajan Tiruchinapalli, David Sidransky, Elizabeth M. Bruckheimer. Development of an androgen-dependent prostate Champions TumorGraft™ cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2793. doi:10.1158/1538-7445.AM2013-2793

Abstract 2205: Integrated next generation sequencing and patient-derived xenografts to personalized cancer treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The knowledge of actionable somatic genomic alterations present in human tumors is enabling the new era of personalized cancer treatment. The great intellectual challenge lies in linking confirmed mutations to protein function. Personalized tumor graft models (Avatars) can aid in the process of genomic analyses interpretation to ultimately move from molecular profile to medication. Methods: Using massive parallel sequencing we performed whole exome sequencing analysis of tumor and matched normal blood samples of 23 patients (pts) with advanced solid tumors (7 lung cancer, 7 pancreatic cancer, 1 neuroendocrine tumor, 2 glioblastoma, 1 uveal melanoma, 2 melanomas and 3 colon cancer) to identify putatively actionable tumor-specific genomic alterations. Avatar models generated by direct engraftment of tumor samples from the pts into immunocompromised mice were used as an in vivo platform to test proposed treatment strategies. Results: Successful exome sequencing analyses has been obtained for 21 pts (1 patient died prematurely, 1 sample was insufficient). Tumor specific mutations (Muts) and copy number variations were identified ranging from 5 to 952 and 0 to 36 respectively. All samples profiled contained clinically meaningful genomic alterations. A successful Avatar model was generated for 10 out of 17 pts. Two engraftment failures corresponded to EGFR mutant lung tumors resected while pts were receiving erlotinib, which initially grew but then regressed. Some of the most relevant drugabble alterations were: KRAS, CHEK1, FGFR2, IGF1R, MET, BRCA1, XPC, NOTCH, CREB3LB, GNA11, SMAD4 and EGFR. In occasions druggable alterations such as muts in NF1, PTPRC, PI3KA and DDR2 failed to provide any benefit when a targeted drug was tested in the Avatar and accordingly treatment of the pts with these drugs was not effective. In one case, loss of STK11 lead to testing of mTOR and SRC inhibitors resulting in tumor regression both in the Avatar and in the clinic. At present time 10 pts have received a personalized treatment: 2 pts, as expected based on the Avatar model, did not response; 4 pts resulted in durable partial remissions and 4 pts are currently on treatment with disease stabilization. In one of the EGFR mutant lung pts the genomic analysis revealed traces of an acquired mutation and allowed decision making at an earlier time point, prior to relapse. Overall, there was a remarkable correlation between drug activity in the Avatar and clinical outcome in the pts, in terms of drug resistance and sensitivity. Conclusion: The detection of actionable tumor-specific genomic alterations in the clinical setting is feasible. However predicting treatment response to known oncogenes is complex and requires detailed information of how different genetic backgrounds function. Avatar models are a powerful investigational platform for therapeutic decision making and help to guide cancer treatment in the clinic. Citation Format: Elena Garralda, Keren Paz, Pedro P. Lopez-Casas, Siân Jones, Amanda Katz, Lisa M. Kann, Fernando Lopez-Rios, Francesca Sarno, Fatima Al-Shahrour, David Vasquez, Elizabeth Bruckheimer, Samuel V. Angiuoli, Luis A. Diaz, Alfonso Valencia, Victor E. Velculescu, David Sidransky, Manuel Hidalgo. Integrated next generation sequencing and patient-derived xenografts to personalized cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2205. doi:10.1158/1538-7445.AM2013-2205

Abstract 1329: Personalizing gastrointestinal cancer treatment by the utilization of TumorGraft™ technology

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignancies of the lower gastrointestinal (GI) tract share similarities in their symptoms, histology, genetic composition and sites of metastasis, but differ in their incidence rates. While colorectal cancer is common with over 130,000 new cases annually in the US alone, small intestinal adenocarcinoma is relatively rare with only 2500 new cases annually. Nevertheless, the overall clinical benefit of “standard of care” therapeutics is negligible, thereby underlining the need for novel therapeutics, especially personalized approaches. Champions Oncology focuses on personalizing cancer treatment via generating Tumorgraft models from each patients individual whole tumor. The current study describes the attempt to identify a personalized solution for two patients with metastatic colorectal and duodenal cancers. Using Champions revolutionary Tumorgraft™ technology, personalized ‘trials’ were performed in vivo. Tumor fragments, obtained from the patients liver metastases, were implanted into immunodeficient mice in a manner that preserves the biological properties of the original tumor and supporting stroma. Once the Tumorgrafts models were established, individual therapeutics and combinations were evaluated to identify treatment regimens likely to be maximally effective in the clinic. Each drug study encompassed 16 different treatment groups, including FDA-approved, as well as investigational agents. Of the different treatment arms in the colorectal model, two demonstrated partial response with tumor growth inhibition values of 152 and 149% for irinotecan/cetuximab/sunitinib and bevacizumab/cetuximab/irinotecan, respectively. While the combination of cetuximab/irinotecan resulted in stable disease in the duodenal model as well, complete responses were observed in all mice treated with eribulin as a single agent. Mutation status analysis of the tumors revealed wild type KRAS. Additional molecular characterization is ongoing to determine other potential signatures of response. The results from the Tumorgraft study were recommended to the patients and their medical oncologists and new lines of treatment were commenced subsequently. The results from these Tumorgraft studies, the molecular profile of the tumors, and correlation with clinical outcome will be presented. Results demonstrate the application of Personalized Tumorgraft models to personalized oncology and their impact on the future of cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1329. doi:1538-7445.AM2012-1329

Abstract 5252: Utilization of TumorGraft technology for personalized breast cancer treatment

Breast cancer is the most common cancer among women with over 280,000 new annual cases and 40,000 deaths in the US alone. While hormonal-dependent breast cancer is correlated with good prognosis, an adequate solution for hormonal-independent tumors remains an unmet medical need, largely due to a significant heterogeneity within tumors and their makeover. Various personalized oncology approaches aim at tailoring the optimal regimen for these patients by better understanding their tumor distinctiveness. Champions Oncology focuses on personalizing cancer treatment through the generation of TumorGraft™ models form the patient9s own tumor. The current study is focused on the attempt to identify a personalized solution for a patient with HER2-amplified metastatic breast cancer whose disease progressed on Trastuzimab (Herceptin). Using Champions novel and cutting-edge TumorGraft™ technology, the challenge was confronted empirically by preforming a personalized clinical trial in vivo. Tumor fragments, obtained from both the primary breast tumor and lung metastasis, were implanted in immune-deficient mice in a manner that preserves the biological properties of the original tumor and its supporting stroma. Once the TumorGraft™ models were established, panel of drugs and drug combinations were evaluated to help identify treatment regimens that are likely to be most effective for the patient clinically. The drug study encompassed 7 different treatment groups including FDA-approved, as well as investigational agents. Of the 7 treatment arms, 3 demonstrated a significant anti-tumor response with tumor growth inhibition values of 80, 93 and 97% for Lapatinib+Capecitabine, Gemcitabine+Carboplatin and Ixabepilone+Capecitabine, respectively. Molecular analysis of the tumor revealed a truncated HER2 (p95) and a robust HER2 translocation to the nucleus. Additional molecular characterization is ongoing to determine other potential signatures of response. Results from the TumorGraft™ study were recommended to the patient and her medical oncologist and new lines of treatment were commenced subsequently. Herein, we describe the data from the TumorGraft™ drug study, the molecular exploration and the pathway analysis of the primary and the metastatic tumor and the consequences correlation to the clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5252. doi:1538-7445.AM2012-5252

Abstract 3641: Breaking through the ceiling: A program to significantly increase efficacy in KRAS and BRAF mutant colorectal cancer xenograft tumors whose growth is arrested by Cetuximab + irinotecan therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cetuximab has significant clinical benefits alone or in combination with irinotecan in metastatic colorectal cancer (mCRC) patients with KRAS wild type tumors, but efficacy in patients with KRAS mutant tumors has thus far been elusive. Mutant BRAF in mCRC tumors may also predict for reduced benefit with EGFR antibodies. Interestingly, in preclinical CRC models, cetuximab, alone or in combination with irinotecan, has significant efficacy in tumor models with mutant KRAS or BRAF. Here we examine if this preclinical efficacy can be significantly increased in a screen for add-on therapies that may provide clinically meaningful efficacy in mCRC patients requiring an EGFR antibody. HT-29 (BRAF mutant-V600E) and HCT-116 (KRAS mutant-G38D) cell lines were utilized as models of CRC. We screened 14 compounds that could potentially add to the effects of cetuximab+irinotecan (C+I) treatment, including inhibitors of inflammation, enzymes, receptor tyrosine kinases, HSP90, cyclins, mTOR, an ATP mimetic, activators of apoptosis and PPARγ. Of the compounds evaluated only 17-AAG (HSP90 inhibitor), everolimus(mTOR inhibitor) and DC101 (antibody targeting murine VEGFR2) significantly broke the ceiling of tumor stasis established with C+I treatment alone in both models. 17-AAG+C+I resulted in T/C% values of 14 and 32% in HCT-116 and HT-29, respectively, compared to 25 and 62% in the C+I treatment group. Follow up studies showed this benefit to be due to an additive interaction between 17-AAG and irinotecan, which was also associated with significant weight loss. Everolimus+C+I treatment resulted in a T/C% of 15 and 22% in HCT-116 and HT-29, respectively, compared to 22 and 35% in the C+I treatment group. The benefits of everolimus+C+I on the other hand required all three agents. The combination of DC101+C+I was significantly more efficacious than C+I in both models, with a T/C% of 11 and 33% in HCT-116 and HT-29, respectively, compared to 19 and 53% in C+I treatment group. In contrast to these three therapies, all other compounds evaluated did not improve the antitumor effects of C+I alone. Results demonstrate a ceiling effect in CRC models with KRAS or BRAF gene mutations that may be associated with the limited clinical benefits observed with cetuximab in patients. The fact that this ceiling can be broken with everolimus in a manner that requires cetuximab, supports the potential utility of cetuximab in these patients in a combination approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2011-3641

Abstract 1187: The role of PI3K as a biomarker

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated in tumorigenesis. Evidence over the past years suggests a pivotal role for the catalytic subunit, PIK3CA, in human cancers. Receptor tyrosine kinases (RTKs) on the cell surface are key regulators of PI3K activity, and RTK antagonists have demonstrated significant anticancer effects in animal models and in patients. However, among tumor harboring activating mutations of PIK3CA, the therapeutic value of RTK antagonists may be compromised. The mutation status of PIK3CA may, therefore, serve as a potential biomarker for both prognosis and response to RTK inhibitors. Somatic mutations of the pik3ca gene occur primarily in three hot spots, E542K, E545K (exon 9) and H1047R (exon 20). These mutations are detected in a broad spectrum of cancer indications including breast, colorectal and lung. This study was designed to explore the nature of PIK3CA mutations among numerous human cancer cell lines, utilizing mostly cells with the capacity to form tumors in mice. Primers corresponding to exon 9 and 20 of human pik3ca gene were designed. Genomic DNA was harvested from over 200 lines, derived from different human cancer indications. The pik3ca locus was sequenced and its WT or mutation status was determined. Of these cells, 8% presented mutations equally distributed between exon 9 and 20. The frequency of PIK3CA mutations was particularly high among colon cancer lines, with fewer incidences among lung, breast and ovarian cancer lines. Interestingly, 50% of the PIK3CA mutated lines presented a concurrent mutation of either KRAS or BRAF. The capacity of PIK3CA mutation-bearing cell lines to respond to RTK blockers was then evaluated, and the mechanism contributing to such phenomenon was further discussed. These results stress PI3K diagnosis value and its potential role as a valid biomarker for patient susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1187.