Jonathon Golas

Delineation of a Cellular Hierarchy in Lung Cancer Reveals an Oncofetal Antigen Expressed on Tumor-Initiating Cells

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.

Control of mammary tumor differentiation by SKI-606 (bosutinib)

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

p120 catenin is a key effector of a Ras-PKCɛ oncogenic signaling axis

Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCɛ) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCɛ in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCɛ in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCɛ in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCɛ-dependent manner, and that loss of PKCɛ signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCɛ is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCɛ ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCɛ as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S‐adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)‐specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met‐cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co‐regulated in models of cancer cell differentiation and co‐expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small‐molecule inhibition leads to de‐repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1‐EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA‐mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Abstract 2113: Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprised of the HER2-targeting antibody trastuzumab and DM1, a microtubule depolymerizing agent covalently attached via a non-cleavable thioether linker. T-DM1 has demonstrated clinical benefit for patients with metastatic breast cancer, however activity may be limited by inherent or acquired resistance during prolonged treatment periods. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 positive tumors, are not well understood. We used the HER2+ cell line N87 to develop a model of T-DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1 resistant population of N87 cells (N87-TM) was generated. N87-TM cells displayed 103-fold resistance toward T-DM1 treatment compared to the parental N87 cells. The N87-TM cells were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug efflux pump (e.g. MDR1) protein expression compared to parental N87 cells. Comparative proteomic profiling suggested an enrichment in proteins (e.g. caveolin-1, CAV1) that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize ADCs into intracellular CAV1+ puncta and alter their trafficking to the lysosome compared to N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached via an enzymatically cleavable linker (i.e. ValCit linker) overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice in vivo formed T-DM1 refractory tumors which remain sensitive to T-ADCs with cleavable-linked auristatin payloads. When comparing antigen positive patient-derived xenograft models that were refractory to T-DM1 yet responded to T-ADCs with ValCit linker-payloads, CAV1 was found to be a predictive protein biomarker identifying T-DM1 refractory tumors. These data implicate caveolae-mediated endocytosis in ADC biology and suggest that alterations in this pathway may impact a tumor9s response profile to ADCs with non-cleavable linkers. We also propose CAV1 as a novel protein biomarker whose high tumoral expression predicts a refractory response to the T-DM1 ADC. Citation Format: Matthew S. Sung, Xingzhi Tan, Christine Hosselet, Michael Cinque, Erik Upeslacis, Jonathon Golas, Fang Wang, Bingwen Lu, Laurie Tylaska, Lindsay King, Jeremy Myers, Edward Rosfjord, Judy Lucas, Hans-Peter Gerber, Frank Loganzo. Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2113.

Abstract B010: Pharmacodynamics and mechanisms of drug action for bispecific redirected T cell immunotherapy against P-cadherin

Utilizing pharmacodynamic immunohistochemistry (PD-IHC) for in situ & quantitative measures, we explored the mechanism of action of a Dual-Affinity Re-Targeting (DART®) bispecific recombinant antibody engineered with enhanced pharmacokinetic properties to extend in vivo half-life. This bispecific, designated P-cadherin LP-DART, is designed to engage and activate polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. Following administration in mice bearing established human tumors and implanted with human T-cells we examined the localization of P-cadherin LP-DART within the tumor xenografts, P-cadherin expression, quantitation and immunophenotyping of tumor infiltrating lymphocytes (TILs), downstream biomarkers of T-cell effector function and immunoregulatory mechanisms. Cell surface P-cadherin expression was maintained on the established tumor xenografts after the administration of single and multiple doses of the bispecific molecule. Furthermore, we detected P-cadherin LP-DART in the tumors more than one week after administration. Pan lymphocyte IHC and digital image analysis demonstrated P-cadherin LP-DART mediated CD3+ T-cell infiltration, resulting in nearly half of the viable cells in the tumor being TILs. Conversely, we did not detect infiltrating CD3+ human T-cells in normal organs, confirming a specific target mediated T-cell response at the tumor site. Elevated proximal and downstream mediators of drug action (granzyme B and cleaved caspase 3) further support that P-cadherin LP-DART localized within the tumor induces T-cell mediated growth inhibition and sustained regression. Additionally, to study the the tumor response to redirected T-cell mediated killing we measured the up-regulation of critical immune check point pathways after treatment P-cadherin LP-DART. In the in vivo tumor models examined, tumor cells acutely and robustly induced expression of immunoregulatory pathways in response to effector T-cell activity. Taken together, we demonstrate the utility of in situ kinetic PD-IHC methodologies to demonstrate target expression, drug localization, downstream biomarkers of drug action, and provide insights into potential immunoregulatory mechanisms in response to T-cell mediated bispecific immunotherapy. Citation Format: Justin Lucas, Andrea T. Hooper, Jonathon Golas, Bryan Peano, Alan Opsahl, Leslie Obert, Maria Gavriil, Timothy Fisher, Anton Xavier, Michael Cinque, Roger Conant, Judy Lucas, Adam Root, Lioudmila Tchistiakova, Hans Peter Gerber, Chad May. Pharmacodynamics and mechanisms of drug action for bispecific redirected T cell immunotherapy against P-cadherin. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B010.

Abstract 564: The expression of fetal oncogene 5T4 in CTCs obtained from NSCLC patients is discordant with the expression measured in the primary tumor

The fetal oncogene 5T4 is a cell surface protein, with over-expression observed in a variety of cancers as compared to normal adult tissue. Recent studies have shown that expression of 5T4 appears to be associated with the undifferentiated state and the epithelial-mesenchymal transition (EMT), and thus has been associated with a more invasive phenotype. We have developed assays to measure the expression of the fetal oncogene, 5T4, in both the primary tumor compartment and in the circulating tumor cell compartment. These assays were then used to investigate 5T4 expression in a small cohort of patients with NSCLC. We obtained matched primary tumor and blood samples, with the blood being obtained prior to resection of the primary tumor. The expression of 5T4 was found to be robust and measurable in both the primary and circulating tumor compartments. We observed expression of 5T4 in both adenocarcinoma and squamous cell carcinoma, in all stages and grades of tumor, with no specific correlation between expression and stage, grade or pathology. We further observed robust enumeration of CTCs in NSCLC samples. The expression of 5T4 was heterogeneous in the CTC compartment with no correlation to grade, stage or pathology. Finally, we observed no concordance between 5T4 expression in the primary tumor and the circulating tumor cell compartment. We discuss the current utility of target expression in predicting response to targeted therapy in the context of antibody based therapy, and the role that CTCs may have in the clinic. Citation Format: Steven R. Pirie-Shepherd, Shibing Deng, Jonathon Golas, Pamela Vizcarra, Eric Tucker, Dena Marrinuci, Hans-Peter Gerber, Eric L. Powell. The expression of fetal oncogene 5T4 in CTCs obtained from NSCLC patients is discordant with the expression measured in the primary tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 564. doi:10.1158/1538-7445.AM2015-564

Abstract 1469: Patient derived xenograft (PDX) models: improving predictability of experimental cancer therapies

Clinical development of cancer therapies is associated with attrition rates as high as 80-95%. This high attrition suggests that standard preclinical pharmacology models do not accurately reflect clinical responses. The development of more predictive preclinical models requires several considerations; the relevance of the in vivo model, the administration of test agent, and the interpretation of efficacy data. PDX are cancer models developed from the direct transfer of patient tumor tissue into immunocompromised mice. A collection of PDX models, by retaining the genetic and histologic characteristics of the patients from which they were derived, represents the complexity and heterogeneity of human cancer. To minimize the clinical attrition rates of oncology compounds, we are developing hundreds of PDX models in seven major cancer indications. The collection is being molecularly profiled by RNAseq, WES, and proteomics. Profiling has identified models with robust expression of target proteins or mutant oncogenes that are likely to respond in preclinical efficacy tests. Conversely, the PDX models may provide an understanding of resistance, for example evaluating models with good target expression that fail to respond to therapy. Patient and tumor information, if known, has been collected for each PDX model including age, sex, cancer stage and grade, diagnosis, primary or metastatic site, and prior treatments. In addition to the improvements provided by the PDX models, a preclinical paradigm shift away from treatment with maximally tolerated dose towards clinically relevant dose (CRD), taking into consideration such aspects as exposure, formulation, route and schedule, is critical when attempting to predict clinical outcome from preclinical data. Also essential is the incorporation of clinically meaningful endpoints (regression) when assessing preclinical activity. We have initiated studies on cohorts of non small cell lung and breast PDX models to predict the likely clinical efficacy of candidate compounds for clinical development and to determine the CRD for standard of care (SOC) regimens required to define the most promising Phase II/III combination therapies. Anti-tumor activities were characterized using RECIST criteria of progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR). Target expression was evaluated by RNA, proteomics and immunohistochemistry. Preliminary results demonstrate a spectrum of responses against experimental therapeutics, including Phase I ADCs and are defining the CRD required for combination treatments with SOC. Identification of the most critical parameters of PDX models predicting clinical outcome will help in validating the utility of ‘n of 1′ studies with the PDX collection, inform patient enrollment strategies, guide combination therapies, and provide insight for identifying new tumor indications. Citation Format: Edward Rosfjord, Xin Han, Danielle Leahy, Erik Upeslacis, Justin Lucas, Jonathon Golas, Andrea Hooper, Fred Immermann, Bingwen Lu, Jeremy Myers, Zhengyan Kan, James Hardwick, Eric Powell, Puja Sapra, Paul Rejto, Hans-Peter Gerber, Judy Lucas. Patient derived xenograft (PDX) models: improving predictability of experimental cancer therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2015-1469

Abstract 5144: Epigenetic reprogramming by tumor-derived EZH2 gain of function mutants leads to aggressive 3D-cell morphologies in both epithelial and melanoma cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In addition to numerous genetic changes underlying cellular transformation, cancer cells are also characterized by epigenetic changes that are likely to play important roles in disease progression. EZH2 is an epigenetic repressor that plays well-established roles in development. In addition to widespread overexpression in a variety of tumors, the discovery of gain of function (GOF) mutations of EZH2 in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and melanoma strongly suggests an important function for this histone methyltransferase in cancer. To ascertain the function of elevated EZH2 catalytic activity, we expressed either wild-type EZH2 (WT) or EZH2 GOF mutants in both non-tumorigenic (immortalized epithelial cells) and tumorigenic (melanoma cells) settings. In both systems, EZH2 GOF mutants greatly increased global levels of H3K27me3 and decreased H3K27me2 levels, similar to the epigenetic pattern seen in DLBCL cell lines with endogenous EZH2 GOF mutations. In epithelial cells, expression of an EZH2 GOF mutant caused striking changes in 3D-morphology and gene changes that are indicative of cells that have undergone an epithelial to mesenchymal transition. In the disease relevant melanoma cells, several distinct EZH2 GOF mutants (but not EZH2 WT) caused prominent branching morphology in 3D-culture. Interestingly, these GOF mutants did not affect 2D-cell morphology or proliferation of melanoma cells. Furthermore, catalytic inhibition of EZH2 GOF mutants with a commercially available tool compound attenuated the 3D-phenotype. Importantly, EZH2 inhibition in melanoma cells expressing an endogenous GOF mutation also caused similar changes in 3D-morphology. RNA-seq analysis revealed genes involved in processes such as cell adhesion and axonal guidance that were down-regulated by EZH2 GOF mutants. Finally, melanoma cells expressing ectopic EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. Collectively, these results suggest that EZH2 GOF mutants may alter the interaction of tumor cells with their microenvironment and in this way provide a selective advantage to such tumors. Citation Format: Robert A. Rollins, Anthony M. Barsotti, Michael Ryskin, Wenyan Zhong, Wei-Guo Zhang, Andreas Giannakou, Christine Loreth, Veronica Diesl, Maximillian T. Follettie, Jonathon Golas, Michelle Lee, Timothy Nichols, Conglin Fan, Gary Li, Stephen Dann, Paul A. Rejto, Kim T. Arndt, Dominique Verhelle. Epigenetic reprogramming by tumor-derived EZH2 gain of function mutants leads to aggressive 3D-cell morphologies in both epithelial and melanoma cells. [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 5144. doi:10.1158/1538-7445.AM2014-5144

Abstract 2669: Targeting the tumor vasculature with antibody drug conjugates

Clinical use of vascular targeting antibody drug conjugates (VT-ADCs) has not yet been explored. Herein, we preclinically demonstrate utility, mechanisms and advantages of VT-ADCs as targeted cancer therapeutics. A monoclonal human IgG 1 antibody selective for murine VEGFR2 (B3) is non-VEGF-A neutralizing and traffics to the lysosome. B3 was conjugated to a proprietary auristatin microtubule inhibitor with a non-cleavable maleimidocaproyl linker, resulting in the proof-of-concept VT-ADC, B3-mcMTI. In vitro, free-payload and anti-VEGFR2-mcMTI selectively inhibit proliferating endothelial cells (ECs) with pM and low nM IC 50 s - suggesting that even with a broadly expressed, low-level target, a VT-ADC should be potent and tumor specific. To test this in vivo, we treated three cell line xenografts (CLX, HT29, Ls174T, A498) and a colorectal carcinoma patient derived xenograft (CRC PDX) with B3-mcMTI at 3 mg/kg q4d, resulting in tumor stasis in four models. In the HT29 colon carcinoma CLX and the CRC PDX model, B3-mcMTI was superior to VEGF-neutralizing antibody G6-31 (anti-VEGF mAb). In standard-of-care (SOC) chemotherapy (irinotecan and 5-FU) combination studies in HT29, B3-mcMTI had improved anti-tumor activity over SOC alone, and, moreover, was as effective in inhibiting tumor growth as a single agent ADC as anti-VEGF mAb combined with SOC. In the 4T1 orthotopic anti-VEGF resistant breast carcinoma setting, B3-mcMTI outperformed anti-VEGF mAb, and when combined with SOC (paclitaxel) demonstrated improved activity over single agent without an increase in metastases. B3-mcMTI internalization in endothelial cells is VEGFR2-mediated, with antibody binding and active payload releasing in normal and tumor tissues at concentrations above the in vitro IC 50 s; however, payload activity as measured by quantitative image analysis of pharmacodynamic biomarkers such as phospho-Histone H3 and cleaved caspase 3 is overwhelmingly localized to tumor ECs. Targeted vessels are smooth muscle invested, suggesting that VT-ADCs may target vasculature that contributes to anti-VEGF resistance. In conclusion, VT-ADCs are effective both as single agents and when combined with SOC, and VT-ADCs may overcome resistance mechanisms to standard anti-angiogenics. Taken together, these data suggest that payloads selective for proliferating ECs enable ADC-mediated targeting of widely expressed EC surface proteins, supporting the clinical pursuit of VT-ADCs. Citation Format: Andrea T. Hooper, Chao-Pei Betty Chang, Kimberly Marquette, Jonathon Golas, Justin Lucas, Timothy Nichols, Judy Lucas, Gavriil Maria, Edward Rosfjord, Anton Xavier, Nathan Scott, Sadhana Jain, Wei Cao, Mauricio Leal, Andreas Maderna, Magali Guffroy, Xiang Zheng, Lioudmila Tchistiakova, Frank Loganzo, Hans-Peter Gerber, Chad May. Targeting the tumor vasculature with antibody drug conjugates. [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 2669. doi:10.1158/1538-7445.AM2014-2669