Heather Hirsch

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclibs antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Evaluating TBK1 as a Therapeutic Target in Cancers with Activated IRF3

TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg. Mol Cancer Res; 12(7); 1055–66. ©2014 AACR. Visual Overview

Phase I Trial of the Human Double Minute 2 Inhibitor MK-8242 in Patients With Advanced Solid Tumors.

Purpose To evaluate MK-8242 in patients with wild-type TP53 advanced solid tumors. Patients and Methods MK-8242 was administered orally twice a day on days 1 to 7 in 21-day cycles. The recommended phase II dose (RP2D) was determined on the basis of safety, tolerability, pharmacokinetics (PK), and by mRNA expression of the p53 target gene pleckstrin homology-like domain, family A, member 3 ( PHLDA3). Other objectives were to characterize the PK/pharmacodynamic (PD) relationship, correlate biomarkers with response, and assess tumor response. Results Forty-seven patients received MK-8242 across eight doses that ranged from 60 to 500 mg. Initially, six patients developed dose-limiting toxicities (DLTs): grade (G) 2 nausea at 120 mg; G3 fatigue at 250 mg; G2 nausea and G4 thrombocytopenia at 350 mg; and G3 vomiting and G3 diarrhea at 500 mg. DLT criteria were revised to permit management of GI toxicities. Dosing was resumed at 400 mg, and four additional DLTs were observed: G4 neutropenia and G4 thrombocytopenia at 400 mg and G4 thrombocytopenia (two patients) at 500 mg. Other drug-related G3 and G4 events included anemia, leukopenia, pancytopenia, nausea, hyperbilirubinemia, hypophosphatemia, and anorexia. On the basis of safety, tolerability, PK, and PD, the RP2D was established at 400 mg (15 evaluable patients experienced two DLTs). PK for 400 mg (day 7) showed Cmax 3.07 μM, Tmax 3.0 hours, t1/2 (half-life) 6.6 hours, CL/F (apparent clearance) 28.9 L/h, and Vd/F (apparent volume) 274 L. Blood PHLDA3 mRNA expression correlated with drug exposure ( R2 = 0.68; P < .001). In 41 patients with postbaseline scans, three patients with liposarcoma achieved a partial response (at 250, 400, and 500 mg), 31 showed stable disease, and eight had progressive disease. In total, 27 patients with liposarcoma had a median progression-free survival of 237 days. Conclusion At the RP2D of 400 mg twice a day, MK-8242 activated the p53 pathway with an acceptable safety and tolerability profile. The observed clinical activity (partial response and prolonged progression-free survival) provides an impetus for further study of HDM2 inhibitors in liposarcoma.

Abstract 3063: Mcl-1 dependency is a predictive biomarker for apoptotic induction by short-term dinaciclib (SCH 727965) treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Dinaciclib (SCH 727965) is a potent and selective inhibitor of the cyclin-dependent kinases (CDKs) 1, 2, 5 and 9 undergoing clinical testing against a range of solid and hematologic malignancies. From preclinical studies, more than 140 cell lines have been profiled for dinaciclib response in long-term (>72 hrs) viability or clonogenicity assays with >97% of the lines showing IC50 β25 nM. This uniformly low nM potency is likely due to repression of both cell cycle progression and transcription through inhibition of CDK1/2 and CDK9, respectively. CDK9 phosphorylation of the RNA pol II (RNAPII) at Ser2 and 5 is required for transcriptional initiation and elongation. We and others have observed rapid CDK9-dependent effects on cells after short-term dinaciclib exposure, including loss of RNAPII Ser2 phosphorylation followed by rapid elimination of the short half-life, pro-survival protein Mcl-1. Since a cancer cells ability to avoid apoptosis is dependent on the balance of several Bcl-2 antiapoptotic family members, including Bcl-2, Bcl-xL and Mcl-1, we hypothesized that Mcl-1 dependent cell lines would be more sensitive to dinaciclib treatment. Moreover, we anticipated that this differential sensitivity could be discriminated from longer-term inhibitory cell cycle effects by conducting short-term dinaciclib exposure assays. Here we report the activity of dinaciclib to induce apoptosis on a panel of 25 human solid tumor cell lines with varying levels of Mcl-1 dependency. Mcl-1 dependency in solid tumor cell lines has been reported to correlate with the Mcl-1 to Bcl-xL mRNA ratio or the level of Mcl-1 gene amplification. Cell viability was assessed after an 18 hour, 100 nM dinaciclib treatment while target engagement and induction of apoptosis was determined after 8 hours. With one exception, all cell lines showed potent CDK9 target engagement as determined by loss of RNAPII Ser2 phosphorylation and corresponding reduced Mcl-1 protein levels. We observed that loss of cell viability, measured by ATP content, directly correlated with the Mcl-1/Bcl-xL mRNA ratio. A dramatic increase in PARP cleavage was also observed in cell lines with the highest Mcl-1/Bcl-xL mRNA ratio. Furthermore, the extent of PARP cleavage correlated with levels of caspase-3/7 activity. Bcl-2 levels did not significantly impact the dinaciclib response. These data provide a rationale for utilizing Mcl-1 dependency as a predictive biomarker for dinaciclib anti-cancer response in solid tumor malignancies. 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 3063. doi:1538-7445.AM2012-3063

Preclinical to clinical translation of anti-PD-1 blockade

Meeting abstracts Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. Immunotherapy with anti–PD-1 monoclonal antibodies such as pembrolizumab shows robust,

Abstract 269: Evaluation of the antitumor activity of anti-PD-1 immunotherapy as a single agent and in combination with approved agents in preclinical tumor models

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid the mechanistic understanding of anti-PD-1 immunotherapy, we generated a surrogate PD-1-blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Gene and protein expression signatures revealed that the more responsive models expressed higher levels of both PD-1 ligand (PD-L1) and tumor-infiltrating lymphocytes compared with nonresponsive models. To further evaluate mechanisms that could potentially enhance the antitumor activity of anti-PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. Because immune suppression is a common side effect associated with many standard-of-care therapies, we evaluated the potential abrogation of muDX400-mediated antitumor activity when combined with approved therapies by scheduling the dosing regimen to examine concurrent and sequential administration of these agents. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by immuno-phenotyping and molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Elaine M. Pinheiro, Ruban Mangadu, Uyen T. Phan, Mingmei Cai, Yanhong Ma, Heather A. Hirsch, Terrill K. McClanahan, Raymond J. Moniz, Ali-Samer Al-Assaad, Samik Basu, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Brian J. Long. Evaluation of the antitumor activity of anti-PD-1 immunotherapy as a single agent and in combination with approved agents in preclinical tumor models. [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 269. doi:10.1158/1538-7445.AM2015-269

Abstract 1307: Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475)

Pembrolizumab (MK-3475) is a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1) that is currently being studied in clinical trials across more than 30 types of cancer. Gene expression profiling was used to explore a less invasive method for gaining new insights into the pembrolizumab mechanism of action and to assess the potential for blood-based predictive biomarkers. RNA-seq data were obtained from the whole blood of 44 patients with advanced melanoma enrolled in the phase 1 KEYNOTE-001 clinical study before and after the first cycle of treatment with pembrolizumab. Objective response rates (ORR) were assessed per RECIST v1.1 by independent central review. The pembrolizumab dose and treatment schedule varied among the patients studied and included 10 mg/kg given once every 3 weeks (Q3W) (n = 21), 10 mg/kg Q2W (n = 12), and 2 mg/kg Q3W (n = 11). Among the 44 patients with melanoma included in the analysis, 75% received previous ipilimumab treatment. The ORR was 32%. Significant posttreatment changes in gene expression were confirmed for the target, PD-1, and its key ligand, programmed death ligand 1 (PD-L1). Changes in PD-1 and PD-L1 gene expression did not, however, show a significant association with ORR. Statistically significant posttreatment changes for an interferon-gamma (IFNG)-related 10-gene signature were observed (P Citation Format: Mark D. Ayers, Michael Nebozhyn, Heather A. Hirsch, Razvan Cristescu, Erin E. Murphy, S. Peter Kang, Scot W. Ebbinghaus, Terrill K. McClanahan, Andrey Loboda, Jared K. Lunceford. Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475). [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 1307. doi:10.1158/1538-7445.AM2015-1307

Abstract 1328: Molecular characterization of mouse syngeneic tumor models in response to treatment with anti-PD-1 immunotherapy

Although there are a variety of immune cells localized in the tumor microenvironment, they are generally present in a quiescent state. The goal of cancer immunotherapy is to activate immune effector cells in the tumor microenvironment to recognize tumor cells and elicit an immune response that results in elimination of the tumor. Checkpoint inhibitors and activators that target T-cell receptors provide attractive targets for cancer immunotherapies by either enhancing T-cell activation pathways or inhibiting T-cell checkpoint/anergy pathways. In 2014, the anti-programmed death receptor 1 (PD-1) antibody pembrolizumab (MK-3475) was approved for use in patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Pembrolizumab is currently being explored in multiple clinical trials covering ∼30 different cancer types. A more complete understanding of the mechanism of action and biology associated with both response and resistance to pembrolizumab is critical to better inform future clinical development and to provide insight into the development of additional immuno-oncology therapies. Preclinical mouse syngeneic tumor models have been used extensively to support the clinical development of drugs such as pembrolizumab and to help identify novel targets that have the potential to synergize with anti-PD-1 therapies. We employed a panel of mouse syngeneic tumor models that have been used to assess the efficacy of muDX400, a fully murinized surrogate antibody of pembrolizumab. Tumors from these models were extensively characterized at the molecular and cellular levels at baseline and after muDX400 treatment. Data obtained from multiple experimental sources, including RNA expression, DNA mutation and copy number, fluorescence-activated cell sorting, and immunohistochemistry, have been integrated to inform pembrolizumab mechanisms of action and resistance, pharmacodynamic biomarkers, responder identification, and indication selection. These findings are also being compared to data being obtained from ongoing clinical trials of pembrolizumab to help better understand whether these preclinical models can be translated to the clinic. These preclinical syngeneic tumor models also provide the opportunity to formulate and test specific hypotheses to more completely understand the biology behind the clinical successes currently observed with novel cancer immunotherapies, including anti-PD-1 antibodies such as pembrolizumab. The hypotheses under evaluation include mutational burden, immune cell activation and migration, interferon signaling, antigen presentation (MHC class I and II), and expression of novel targets that will help lead to the development of the next generation of cancer immunotherapies. Citation Format: Heather A. Hirsch, Elaine M. Pinheiro, Mingmei Cai, Yanhong Ma, Manjiri Sathe, Mark Ayers, Terrill K. McClanahan. Molecular characterization of mouse syngeneic tumor models in response to treatment with anti-PD-1 immunotherapy. [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 1328. doi:10.1158/1538-7445.AM2015-1328

Abstract 4714: Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer

MK-4280 is a humanized IgG4 monoclonal antibody (mAb) that binds to the immune checkpoint receptor Lymphocyte Activation Gene-3 (LAG-3) to block the interaction with its ligand, Major Histocompatibility Complex (MHC) Class II. LAG-3 is frequently co-expressed with other immune checkpoint receptors, most notably programmed cell death protein-1 (PD-1), on T cells with an exhausted phenotype. LAG-3 and PD-1 cooperate to regulate peripheral immune tolerance in healthy individuals, and, conversely, play critical roles in several diseases, including autoimmunity, graft rejection, viral infections, and cancer. Co-blockade of LAG-3 and PD-1 in immunocompetent mouse tumor models have demonstrated augmented anti-tumor activity over single agents. However, the molecular mechanisms behind these combination effects have not been fully investigated. Here, preclinical proof-of-biology studies are presented for co-targeting LAG-3 and PD-1 in cancer. c28G10-mG1-[D265A] (abbreviated 28G10-mG1) is a rat:mouse chimera that mimics MK-4280 by its ability to directly block the mouse LAG-3:MHC Class II interaction without initiating Fc-mediated effector functions. As a single agent, 28G10-mG1 demonstrated modest anti-tumor activity across several syngeneic mouse tumor models, despite evidence of systemic drug exposure and target engagement (as assessed by sLAG-3-mAb complex accumulation). The combination of 28G10-mG1 and the anti-mouse PD-1 blocking antibody mDX400 resulted in greater tumor growth inhibition and increased numbers of complete responses (CR) over mDX400 alone in the MBT-2 tumor model. Furthermore, animals that had achieved CR to combination therapy were subsequently protected from MBT-2 re-challenge, suggesting the establishment of immune memory. RT-qPCR analyses revealed up-regulation of immune-related genes, primarily at Day 4 in the blood and Day 8 in the tumor with mDX400, but not 28G10-mG1, treatment. However, when combined with mDX400, 28G10-mG1 further altered the expression of many immune-related genes that were perturbed by mDX400 single agent therapy. Genes unique to combination treatment were also observed. Significantly, immune pathway signatures associated with clinical efficacy to Keytruda were upregulated with combination therapy. Tumor transcriptome and network analysis by RNAseq revealed enrichment in several immune- and cytokine-related pathways with combination therapy compared to mDX400 single agent therapy. Taken together, these preclinical oncology studies support the concept of co-targeting LAG-3 to increase the therapeutic efficacy of PD-1 blockade. Clinical investigation of MK-4280 in combination with anti-PD-1 therapy (pembrolizumab/Keytruda) is ongoing. Citation Format: Brian B. Haines, Sarah Javaid, Long Cui, Heather Hirsch, Saso Cemerski, Terri McClanahan, Manjiri Sathe, Shuli Zhang, Michael Rosenzweig, Brian Long, Rene de Waal Malefyt. Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer [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 4714. doi:10.1158/1538-7445.AM2017-4714

Abstract B114: Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid in the mechanistic understanding of anti–PD-1 immunotherapy, we generated a surrogate PD-1–blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Using a multifaceted approach, tumors from these models were extensively characterized at the molecular and cellular level by gene expression profiling, whole exome sequencing, fluorescence-activated cell sorting, and immunohistochemistry to help elucidate mechanisms of action and biology associated with response and resistance to anti-PD1 treatment. Analyses included but were not limited to the evaluation of mutational burden, immune cell activation and migration, interferon signaling, antigen presentation (major histocompatibility [MHC] class I and II), and expression of novel targets.To further evaluate mechanisms that could potentially enhance the antitumor activity of anti–PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Heather Hirsch, Ruban Mangadu, Mingmei Cai, Yanhong Ma, Uyen Phan, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Terri McClanahan, Brian Long, Elaine M. Pinheiro. Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents. [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 B114.