Igor Feldman

Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors

Phosphorylation sites on proteins in the phosphatidylinositol 3-kinase pathway that are regulated by candidate drugs can serve as useful biomarkers to predict tumor sensitivity to AKT inhibitors. Toward Customizing Tumor Treatment Just as our view of Earth has become increasingly global, cells are now seen as complex networks of interacting and intersecting signaling pathways rather than a collection of regulated genes. This new view applies to cancer cells as well, which we now know have entire dysregulated pathways and not just dysregulated genes. Andersen and colleagues have identified phosphoprotein biomarkers for a pathway often altered in cancer—the phosphatidylinositol 3-kinase (PI3K) pathway—and have shown that one of these predicts the sensitivity of cancer cells to a promising class of cancer drugs: inhibitors of AKT, a kinase that promotes growth and inhibits cell death. To find useful markers of the PI3K pathway, the authors focused on a vital biochemical event—the addition of phosphate groups to serines and threonines in cellular proteins. Cells use this simple covalent modification over and over again to regulate protein-protein binding and activity of key enzymes. Measurement of this modification in specific proteins reveals their activation. The authors monitored 375 phosphorylation sites in the PI3K pathway after treating prostate cancer cells with three different PI3K pathway inhibitors, potentially useful drugs. They found that each drug modulated a specific array of phosphoproteins, with some overlap, many of them within proteins that participate in cytoskeletal remodeling, vesicle transport, and protein translation. In theory, each phosphopeptide that decreased in abundance after drug treatment could, if elevated in cancer cells, serve as a biomarker of sensitivity to that drug. To show that this was the case, the authors chose one of the phosphorylated sites (the threonine at position 246 of the cytoplasmic protein PRAS40) and generated a high-quality antibody to it. The amount of phosphorylation at Thr246 correlated with activation of the PI3K pathway in human cancer cell lines, in a mouse prostate tumor, and in triple-negative breast tumors. Of potentially even more utility, Thr246 phosphorylation predicted the sensitivity of these cells to AKT inhibitors. Cancers are extremely heterogeneous, even within tissues, and for optimal effectiveness, treatments need to be customized accordingly. As this work shows, phosphorylated amino acids can serve as biomarkers for activated pathways in cancer and, because specific antibodies can easily be made to these phosphorylated peptides, can be readily measured. These results point to a way, after further development of more biomarkers, to routinely characterize the activated pathways in patients’ cancers. A tumor characterized in this way can then be treated with the appropriate pathway-specific drugs, optimizing the chances of eradicating the tumor. Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K–mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway–relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40Thr246. Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)–deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40Thr246 positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKTThr308, the phospho-PRAS40Thr246 epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775–induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC50 of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1S345, induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity. Mol Cancer Ther; 12(8); 1442–52. ©2013 AACR.

Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition.

BackgroundInhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models.MethodsSmall molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively.ResultsIn vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model.ConclusionsThese results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

An Unbiased Oncology Compound Screen to Identify Novel Combination Strategies

Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo. This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155–62. ©2016 AACR.

EZH2 Inhibition by Tazemetostat Results in Altered Dependency on B-cell Activation Signaling in DLBCL

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

Abstract 4931: Biomarker discovery in a large panel of cell lines shows different sample size requirement for prediction of response across a set of compounds

A panel of more than 600 cell lines from 17 tumor types has been profiled and sensitivity to a set of FDA approved compounds with different mechanisms of action has been tested. Comparison of gene expression profiles with overlapping set of publicly available profiles showed 100% accuracy of cell line identity prediction using nearest neighbor classifier. Similar analysis of CNV data had 80% accuracy due to relatively little CNV perturbation in some of the cell lines. Significant gene expression signatures have been detected for 80% of compounds. De-novo agnostic classification based on 50% train/test split and a linear classifier resulted in significant prediction on the test set for about 40% of the compounds, such as dasatinib, 5FU, paclitaxel, but failed to produce a significant prediction for others, such as doxorubicine, irinotecan, and vinblastine. For most of the compounds, the prediction of response is complex, with multiple distinct molecular features contributing to a classification algorithm. This inherent complexity requires integration of gene expression, CNV and mutation data as well as a large cell line sets for development of accurate classification algorithms. We defined functional CNV and SNV events using gene expression based modules as a functional readout. Predictive models that incorporate prior knowledge of mechanism of action of the compounds and rely on functional SNV and CNV events out perform completely agnostic methods of prediction. 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 4931. doi:1538-7445.AM2012-4931

Abstract 406: CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships

Target identification is a critical step in drug discovery, but the process has many challenges including non-specific reagents, limited ability to test numerous models, and incomplete target inhibition. Pooled screening with CRISPR/Cas9 permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To determine the specific dependencies of cell lines on epigenetic pathways, we designed a CRISPR/Cas9 library to target 640 epigenetic genes and screened more than 200 cell lines covering a variety of oncology indications, including breast, lung, and renal cell carcinoma (RCC). We find that CRISPR pooled screening is a highly effective approach for target identification and provides robust, highly reproducible data as long as a sufficient number of small guide RNAs are used. We identify known pan-essential genes, including in the transcription (CDK9), translation (EIF4A1 and EIF4A3) and splicing (SRSF2) machinery. We additionally identify many novel pan-essential genes across a variety of epigenetic pathways, including histone acetylases and deacetylases, chromatin remodeling factors, helicases and others. We also investigated epigenetic synthetic lethal interactions that have been previously reported. For example, it has been reported that the SWI/SNF family displays paralog synthetic lethality for SMARCA2 in the context of SMARCA4 mutations, and for ARID1B in the context of ARID1A mutations. While we do see that some of the same trends hold, the synthetic lethal relationship appears to be more complex than previously realized, including the need to examine mRNA levels in addition to mutation type. Most importantly, we identify more than 100 epigenetic genes which show selective sensitivity, i.e. where knockout shows an anti-proliferative effect in only a subset of the cell lines. These are the most promising targets for further drug discovery programs. We have used additionally CRISPR/Cas9-domain based screening to identify the functionally relevant sites for many of these genes. Furthermore, we can overlay gene expression and mutation data to identify novel synthetic lethal relationships. One gene that displays selective sensitivity is EGLN1, the prolyl hydroxylase for the hypoxia-inducible factor, HIF1α. We find that EGLN1 is required for proliferation only in RCC cell lines which retain wild-type VHL, another component of the hypoxia response pathway, which is frequently lost in RCC. As such, EGLN1 loss is synthetically lethal in the presence of wild-type VHL in RCC cells. Thus this approach not only identifies an enzymatic drug target but also a patient stratification method. Other novel synthetic lethal interactions have also been identified. Our data demonstrates that CRISPR pooled screening is a powerful technique for identification of epigenetic synthetic lethal interactions. Citation Format: Alexandra R. Grassian, Darren Harvey, Julian Fowler, Allison E. Drew, Igor Feldman, Richard Chesworth, Robert Copeland, Jesse J. Smith, Scott Ribich. CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2017-406

Abstract 586: Discovery of a novel TIM3 binding partner and a key role for TIM3 on macrophages: Identification of specific antibodies capable of converting immune-suppressive macrophages to immune-enhancing

Our Translational Science Platform uses an unbiased bioinformatics-based approach to interrogate particular cell types within the tumor microenvironment (TME). Given the correlation between high levels of immune-suppressive macrophages within the tumor TME and poor patient prognosis across a number of solid tumor types we focused initially on developing novel immunotherapies to modify this cell type. We identified 10 targets as candidates for converting tumor-associated macrophages from immune-suppressing to immune-enhancing. One of these targets was TIM3. To date, TIM3 has been pursued mainly as a checkpoint target for T cell-directed immunotherapies based on its expression on exhausted T cells. Anti-TIM3 mAbs, generated by multiple groups, induce responsiveness in T cells and demonstrate anti-tumor benefit in certain mouse models. However, our macrophage-centric approach has identified a previously unrecognized protein-protein interaction between TIM3 and one of our additional macrophage targets. Based on knowledge of this interaction, we were able to generate and select for panels of mAbs to TIM3 and to its binding partner capable of converting macrophages from an “M2” to an “M1” pro-inflammatory phenotype. In contrast to published anti-TIM3 mAbs, our particular anti-TIM3 mAbs lacked activity in T cell-based assays, but promoted an increase in pro-inflammatory cytokines with a reduction or no effect in anti-inflammatory cytokines in a macrophage activity assay. In this assay, monocytes were prepared from human peripheral blood and cultured in the presence of M-CSF to bias toward an M2 phenotype. Under sub-optimal stimulation with LPS or CD40L or HMGB1, treatment of these cells with the anti-TIM3 mAbs led to increases in pro-inflammatory cytokines including IL-1β and TNFα. The conversion to an “M1” macrophage by anti-TIM3 mAbs had downstream consequences on T cells as demonstrated by mixed lymphocyte reaction experiments. In these studies, the addition of anti-TIM3 led to a macrophage-dependent increase in IFNγ from the T cells. To assess the impact of our anti-TIM3 mAbs in the tumor setting, tumor histoculture experiments were performed. Tumor tissue slices from ovarian cancer patients treated with anti-TIM3 showed an increase in a range of cytokines and in this tumor setting the initial sub-optimal stimulus was not required. Specific antibodies to TIM3 and its binding partner that are able to promote a pro-inflammatory macrophage phenotype have been generated. We are developing these as modulators of the TME, to be assessed either as single agents or in combination with other therapies such as checkpoint inhibitors. Citation Format: Jamie Wong, Ryan Phennicie, Igor Feldman, Sriram Sathyanarayanan, Don Shaffer, Mohammad Zafari, Steve Sazinsky, Kenneth Crook, Debbie Law. Discovery of a novel TIM3 binding partner and a key role for TIM3 on macrophages: Identification of specific antibodies capable of converting immune-suppressive macrophages to immune-enhancing. [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 586.

Abstract P2-11-07: Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer

B7-H4 (VTCN1, B7x, B7S1) is a transmembrane protein belonging to the B7 family of costimulatory proteins and has been shown to inhibit T cell proliferation, cytokine secretion, and cytotoxic lymphocyte (CTL) induction. B7-H4 expressed on tumor cells or macrophages has been associated with poor prognosis and impaired T cell function in renal cell and ovarian cancers. Here we show B7-H4 is abundantly expressed in human breast cancer with triple negative breast cancer (TNBC) having the highest overall B7-H4 mRNA expression. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluation of B7-H4 protein and quantified B7-H4 expression in 156 breast tumor samples. Approximately 70% of the breast tumor samples had detectable B7-H4 expression whereas none of the normal or benign breast tissues stained positive for B7-H4. Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, the CD45+ immune cells were negative for B7-H4 expression. Interestingly none of the TNBC samples that were positive for B7-H4 showed detectable expression of PD-L1 suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. To evaluate the role of B7-H4 on tumor immune evasion, we overexpressed murine or human B7-H4 on the mouse colon-26 (CT26) tumor cell line and injected these cells intravenously into Balb/c mice. By day 14 we observed significantly more tumors as well as larger percent tumor area in the lungs of mice given CT26 cells transduced with human or mouse B7-H4 as compared to vector control transduced cells. These data suggest B7-H4 expression in tumors can accelerate tumor growth in immune competent mice and that targeting B7-H4 may provide therapeutic benefit. Given the mutually exclusive expression patterns of B7-H4 and PD-L1 a B7-H4 targeting agent may provide particular benefit in those patients where current anti-PD-1/PD-L1 therapies are not effective. Citation Format: Shaffer DR, Nagashima K, Cortez-Retamozo V, Feldman I, Smith J, Zafari M, Larson R, Mabry R, Novorantseva T, Briskin M, Sathyanaryananan S. Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-07.