Clinical Development Of Inhibitors Research Articles

A Phase I/Ib study of MEK162 (binimetinib), a MEK inhibitor, in combination with carboplatin and pemetrexed in patients with non-squamous NSCLC.

TPS2609Background: RAS is the most commonly mutated oncogene in lung cancer. While direct inhibitors of RAS have not yet been successfully developed, MEK inhibitors have shown activity in RAS mutated tumors including KRAS mutant NSCLC. Binimetinib (ARRAY, Boulder CO) is an oral highly selective inhibitor of MEK1/2 and has demonstrated preclinical and clinical activity. Distinct from other MEK inhibitors in clinical development, it has activity against NRAS mutant cancers, with improved PFS as a single agent in NRAS mutant melanoma compared to dacarbazine (NEMO). Methods: In this phase I/Ib study, binimetinib is combined with pemetrexed/carboplatin in advanced non-squamous NSCLC patients. Primary study objectives include determination of the RP2D of continuous binimetinib plus pemetrexed/carboplatin, safety (NCI CTCAE v. 4) of the combination and exploration of efficacy (ORR per RECIST v.1.1). Population PK is being characterized and potential relationships between RAS mutation subtypes and response explor...

Inhibition of Heat Shock Protein 90 Prevents HIV Rebound

HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4+ T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ−/− bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.

Abstract P2-05-17: ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis

Abstract Several lines of experimental evidence suggest that Hepatocyte Growth Factor (HGF) and its receptor MET play an important role in breast cancer progression and drug resistance. To date, targeted MET inhibitors in clinical development have primarily shown cytostatic rather than cytotoxic effects. Development of a cytotoxic MET inhibitor would serve to complement standard breast cancer therapy, especially when administered in the adjuvant/neo-adjuvant setting. We have developed ARGX-111, a human antibody antagonist of MET function. ARGX-111 blocks both HGF-dependent and -independent signaling, down-regulates tumor cell surface expression of MET and kills MET-overexpressing cells by enhanced antibody-dependent cellular cytotoxicity (ADCC). ARGX-111 was shown to be more efficacious than an ADCC-inactive control antibody in both HGF-dependent and -independent tumor xenograft models. ADCC reporter assays confirmed the cytotoxic effects of ARGX-111 in patient-derived primary tumor specimens, including MET-expressing breast cancer stem-like cells. In an orthotopic mouse model of metastatic mammary carcinoma (MDA-MB-231), adjuvant or neo-adjuvant treatment with ARGX-111 was significantly more effective in depleting circulating tumor cells (CTCs) and suppressing the development of bone and lung metastases than the ADCC-inactive control. Taken together, these results provide a rationale for clinical investigation of ARGX-111 in the early breast cancer setting. An ongoing Phase 1 study (NCT02055066) is examining the effects of ARGX-111 on CTCs, alongside the assessment of its safety and efficacy. Citation Format: Morello V, Hultberg A, De Jonge N, Huyghe L, Hanssens V, Brouckaert P, Saunders M, Dreier T, Thibault A, Rolfo C, Aftimos P, Awada A, Michieli P, de Haard H. ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis. [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 P2-05-17.

Recurrent AKT mutations in human cancers: functional consequences and effects on drug sensitivity.

Precision oncology trials based on tumor gene sequencing depend on robust knowledge about the phenotypic consequences of the genetic variants identified in patients' tumors. Mutations in AKT1-3 occur in 3-5% of human cancers. Although a single hotspot mutation, E17K, is the most common, well characterized activating mutations account for a minority of Akt variants that have been identified in large tumor sequencing studies to date. In order to determine the potential clinical relevance of both common and rare Akt mutations, we expressed a set of over twenty recurrent Akt mutants in three different cell lines and evaluated activation of Akt pathway signaling and effects on growth. We determined their relative sensitivity to allosteric and ATP-competitive Akt inhibitors in clinical development. Most Akt mutants did not activate pathway signaling compared to wild type Akt and did not affect growth properties. In addition, the most common activating Akt mutations, including Akt1 E17K, L52R, and Q79K conferred neither sensitivity nor resistance to Akt inhibitors. Equivocal evidence was found that Akt1 D323H and Akt2 W80C mutants are relatively resistant to the allosteric Akt inhibitor MK-2206, but not an ATP-competitive inhibitor. Our results suggest that the vast majority of rare Akt variants are passenger mutations with no effect on drug sensitivity. The hypothesis that activating Akt mutations predict for Akt inhibitor sensitivity remains to be tested clinically, but is not yet supported by our preclinical data.

Abstract PL05-04: PARP trapping versus PARP catalytic inhibition and coupling with TDP1

Abstract The five clinical PARP inhibitors (veliparib, olaparib, niraparib, rucaparib and talazoparib) are potent submicromolar competitive NAD+ inhibitors for PARP1 and PARP2, thereby blocking PARylation reactions [i.e. formation of poly(ADPribose) polymers]. In addition, PARP trapping, which determines the anticancer activity of PARP inhibitor as single agents, is drug-specific, and PARP inhibitors can be ranked according to their PARP trapping potency: talazoparib >> niraparib ≈ olaparib ≈ rucaparib > veliparib. We will review evidence that PARP trapping is the primary cytotoxic mechanism of PARP inhibitors as single agents and that cancer-specific DNA repair alterations as well as SLFN11 expression determine the cytotoxicity of trapped PARP-DNA complexes in cancers beyond BRCA inactivation. Used in combination in cellular models, PARP inhibitors are highly synergistic with alkylating agent (temozolomide or methyl methanesulfonate) and topoisomerase I (Top1) inhibitors (camptothecin and its clinical derivatives (topotecan and irinotecan) and the non-camptothecin indenoisoquinoline Top1 inhibitors in clinical development (LMP400, LMP776 and LMP744). Both alkylating agents and Top1 inhibitors induce DNA single-strand breaks sensed by PARP. Yet, the molecular mechanisms of synergy are different. For alkylating agents (temozolomide and MMS), both PARP trapping and PARylation inhibition account for the synergy, whereas for Top1 inhibitors, there is no involvement of PARP trapping and it is PARylation inhibition that deters the coupling of PARP with the repair enzyme, tyrosyl-DNA phosphodiesterase, TDP1. We will review the molecular pharmacology differences between PARP inhibitors as single agents and the rationale for choosing among different PARP inhibitors in combination with alkylating agents or Top1 inhibitors based on trapping vs. catalytic inhibition. Citation Format: Yves G. Pommier, Junko Murai. PARP trapping versus PARP catalytic inhibition and coupling with TDP1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL05-04.

Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention.

Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3(+) T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

ABT-199 (venetoclax) and BCL-2 inhibitors in clinical development.

With the advent of new agents targeting CD20, Bruton’s tyrosine kinase, and phosphoinositol-3 kinase for chronic lymphoid leukemia (CLL), more treatment options exist than ever before. B-cell lymphoma-2 (BCL-2) plays a major role in cellular apoptosis and is a druggable target. Small molecule inhibitors of BCL-2 are in active clinical studies. ABT-199 (venetoclax, RG7601, GDC-0199) has been granted breakthrough designation by FDA for relapsed or refractory CLL with 17p deletion. In this review, we summarized the latest clinical development of ABT-199/venetoclax and other novel agents targeting the BCL-2 proteins.

Abstract IA41: Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response.

Abstract The discovery of JAK2/MPL mutations in the majority of patients with Myeloproliferative Neoplasms (MPN) led to the development of JAK kinase inhibitors. JAK kinase inhibitors, including ruxolitinib, improve patient constitutional symptoms and reduce splenomegaly, but do not significantly reduce mutant allele burden. Chronic exposure to JAK kinase inhibitors results in JAK inhibitor persistence in cell lines, murine models, and patient samples, which we have shown is due to JAK2-transactivation and persistent JAK-STAT signaling. We have used murine genetic studies show that MPN cells remain dependent on JAK2 expression for proliferation and survival. These data suggest that pharmacologic approaches that better inhibit JAK2 activity in MPN cells may demonstrate increased efficacy in vivo. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. CHZ868 showed significant activity in murine models of Jak2V617F-induced polycythemia vera, and MPLW515L-mutant MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden in these models. In addition, it has not been delineated whether the clinical benefits of JAK inhibitors are solely from target inhibition in MPN cells, or if JAK-STAT inhibition in malignant and non-malignant cells contributes to the therapeutic response. We show that the levels of circulating cytokines are elevated in MF and that cytokine production is reduced with JAK inhibitor treatment. Importantly, aberrant cytokine production in MF emanates from both malignant and non-malignant cells, and that JAK inhibition reduces cytokine production from both tumor and non-tumor populations. Our data suggest inhibition of JAK-STAT signaling in malignant and non-malignant cells is required to improve symptoms, reduce disease severity, and to reduce disease progression. Moreover, optimizing therapies to potently inhibit JAK-STAT signaling in malignant and non-malignant cells are warranted to improve clinical efficacy and outcomes in MPN patients. Citation Format: Sara Meyer, Maria Kleppe, Neha Bhagwat, Priya Koppikar, Minsuk Kwak, Thomas Radimerski, Rong Fan, Ross L. Levine. Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA41.

Abstract IA08: The microenvironment in CLL: Cellular and molecular players and emerging therapeutic targets.

Abstract The tissue microenvironment plays an important role of in the pathogenesis of chronic lymphocytic leukemia (CLL), delivering signals that promote the clonal expansion of the malignant B cells, as well as drug resistance. The complex cross talk between neoplastic B cells and the microenvironment, for example in secondary lymphoid organs, has been increasingly well characterized, and several cellular and molecular key players have emerged. Important cells in the CLL microenvironment are monocyte-derived nurselike cells (NLC), mesenchymal stromal cells (MSC), and T cells, which engage in a dialogue with the neoplastic CLL cells, often via specific receptor-ligand pairs. Engagement of CLL surface receptors by ligands in the tissue microenvironment, for example activation of B cell receptor (BCR) signaling, triggering of TNF family members (i.e. BAFF, APRIL), and activation of chemokine receptors and adhesion molecules result in the activation of signaling cascades that support the maintenance and expansion of the CLL clone. BCR signaling plays a particularly important pathogenic role in CLL, based on structural restrictions of the BCR, and signs of BCR-dependent survival and growth of the malignant B cells. Ligand-independent (“autonomous”) and ligand-dependent BCR signaling have been characterized, the latter triggered by (auto-) antigens present in the tissue microenvironment. Based on high response rates and durable remissions in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR-associated kinases (BTK, PI3Kδ), which currently are changing treatment paradigms in CLL. Clinical and translational aspects of these new targeted therapeutics will be discussed, along with an outlook into future therapeutic strategies. Citation Format: Jan A. Burger. The microenvironment in CLL: Cellular and molecular players and emerging therapeutic targets. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA08.

Abstract 2589: Investigations of covalent binding efficiency for third-generation EGFR irreversible inhibitors AZD9291 and CO-1686

Abstract Patients with non-small cell lung cancer with activating EGFR mutations initially respond to first-generation and second-generation EGFR inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR T790M mutation. In addition, patients oftentimes experience severe side effects including rash and diarrhea due to the inhibition profile against wild-type EGFR. AZD9291 and CO-1686 are the leading third-generation irreversible EGFR inhibitors in clinical development. They selectively inhibit T790M EGFR, while sparing wild-type EGFR. In this study, we investigated the covalent binding efficiency of these two irreversible EGFR inhibitors using biochemical and cellular assays. We discovered that these two inhibitors had dramatic difference in covalent binding efficiencies in WT and mutant EGFR binding at biochemical and cellular levels. In pEGFR cellular assay, AZD9291 displayed much prolonged inhibition of pEGFR than CO-1686. In CO-1686 treated cells, recovery of pEGFR had reached 50% of control levels only after 4 hours. In the meantime, pEGFR in AZD9291 treated cells had no recovery even after 24 hours. These data may suggest different covalent bond formation efficiency and reversibility between different types of irreversible EGFR inhibitors. This may be one of the important factors for consideration when develop third-generation EGFR inhibitors. Citation Format: Zhen Qin, Xuebing Sun, Ye Liu, Yunguang Du, Miao Wang, Nan Hu, Jiye Zhang, Hao Peng, Lai Wang, Min Wei, Lusong Luo. Investigations of covalent binding efficiency for third-generation EGFR irreversible inhibitors AZD9291 and CO-1686. [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 2589. doi:10.1158/1538-7445.AM2015-2589

Abstract 778: The identification and structure-guided optimisation of potent and selective inhibitors of oncogenes in medullary thyroid carcinoma and lung adenocarcinoma

Abstract RET (REarranged during Transfection) is a receptor tyrosine kinase (TK), which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activating mutations in RET (C634W and M918T) have been identified in both familial and sporadic forms of medullary thyroid carcinoma (MTC) and correlate with aggressive disease progression, validating RET as a classical oncogene. Furthermore the recent identification of RET fusions (CCDC6-RET and KIF5B-RET) present in ∼1% of lung adenocarcinoma (LAD) patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been identified opportunistically through selectivity profiling of compounds initially designed to target other TKs. Such “secondary RET inhibitors” include the clinical agents Vandetanib and Cabozantinib, both approved for use in MTC, but additional pharmacological activities (most notably inhibition of KDR) lead to dose-limiting toxicity. Using a robust screening cascade developed in house, we have measured RET and KDR inhibitory activity in vitro and in relevant cell line models to assess compound potency and selectivity. Anti-proliferative activity and off-target toxicity of these agents have also been measured. Although these competitor compounds displayed reasonable RET potency in cellular assays and this translated into anti-proliferative effects in our MTC and LAD disease models, as expected none met our target candidate criteria, clearly highlighting the need for therapeutic agents with improved selectivity. Guided by structure-based drug design, we have identified and optimised a novel series of potent and selective inhibitors of the RET kinase domain. These agents met our stringent criteria for enzyme and cell selectivity and, whilst potent in a RET-driven cell line, display little overt toxicity in a matched non-RET driven cell line. Herein, we describe the chemical optimisation of these agents and, using structural information, rationalise their improved selectivity. Citation Format: Roger J. Butlin, Rebecca Newton, Mandy Watson, Gemma Hopkins, Ben Acton, Kate Bowler, Samantha Fritzl, Kristin Goldberg, Niall Hamilton, Sarah Holt, Stuart Jones, Allan Jordan, Nikki March, Daniel Mould, Helen Small, Alexandra Stowell, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. The identification and structure-guided optimisation of potent and selective inhibitors of oncogenes in medullary thyroid carcinoma and lung adenocarcinoma. [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 778. doi:10.1158/1538-7445.AM2015-778

The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma.

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.

Frequent promoter hypermethylation of PTPRT increases STAT3 activation and sensitivity to STAT3 inhibition in head and neck cancer.

STAT3 overactivation is a common event in many cancers, including head and neck squamous cell carcinoma (HNSCC), where STAT3 represents a promising therapeutic target. HNSCC is not characterized by frequent kinase mutations, in contrast to some malignancies where mutational activation of kinases upstream of STAT3 is common. Instead, STAT3 may be activated by loss-of-function (LOF) of negative regulators of STAT3, including by promoter hypermethylation of PTPRT. Here we first analyzed The Cancer Genome Atlas (TCGA) data and determined that the PTPRT promoter is frequently hypermethylated in several cancers, including HNSCC (60.1% of tumors analyzed) in association with downregulation of PTPRT mRNA expression and upregulation of pSTAT3 expression. These findings were confirmed in an independent cohort of HNSCC tumors by methylation-specific PCR (MSP) and immunohistochemistry. We demonstrate that PTPRT promoter methylation and gene silencing is reversible in HNSCC cells, leading to PTPRT-specific downregulation of pSTAT3 expression. We further show that PTPRT promoter methylation is significantly associated with sensitivity to STAT3 inhibition in HNSCC cells, suggesting that PTPRT promoter methylation may serve as a predictive biomarker for responsiveness to STAT3 inhibitors in clinical development.

Poly (ADP-ribose) polymerase inhibitors: recent advances and future development.

Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising activity in epithelial ovarian cancers, especially relapsed platinum-sensitive high-grade serous disease. Consistent with preclinical studies, ovarian cancers and a number of other solid tumor types occurring in patients with deleterious germline mutations in BRCA1 or BRCA2 seem to be particularly sensitive. However, it is also becoming clear that germline BRCA1/2 mutations are neither necessary nor sufficient for patients to derive benefit from PARP inhibitors. We provide an update on PARP inhibitor clinical development, describe recent advances in our understanding of PARP inhibitor mechanism of action, and discuss current issues in the development of these agents.

O6.1 - Classification of PARP inhibitors based on PARP trapping and catalytic inhibition, and rationale for combinations

ABSTRACT All PARP inhibitors in clinical development (veliparib, olaparib, niraparib, rucaparib, and talazoparib) are potent submicromolar competitive NAD+ inhibitors for PARP1 and PARP2, thereby blocking PARylation reactions [i.e. formation of poly(ADPribose) polymers]. In addition, PARP trapping, which determines the anticancer activity of PARP inhibitor as single agents, is drug-specific, and PARP inhibitors can be ranked according to their PARP trapping potency: talazoparib >> niraparib ≈ olaparib ≈ rucaparib > veliparib. The highly synergistic effects of PARP inhibitors in combination with alkylating agent (temozolomide or methyl methanesulfonate) and topoisomerase I (Top1) inhibitors (camptothecins and indenoisoquinolines) are well documented. Both classes of drugs induce DNA single-strand breaks sensed by PARP. Yet, the molecular mechanisms of synergy are different. For alkylating agents (temozolomide and MMS), both PARP trapping and PARylation inhibition account for the synergy, whereas for Top1 inhibitors, there is no involvement of PARP trapping and it is PARylation inhibition deters the coupling of PARP with the repair enzyme, tyrosyl-DNA phosphodiesterase TDP1. We will review the differences between PARP inhibitors and the rationale for choosing among different PARP inhibitors in combination with alkylating agents or Top1 inhibitors.

Type II Inhibition of JAK2 with NVP-CHZ868 Reverses Type I JAK Inhibitor Persistence and Demonstrates Increased Efficacy in MPN Models

The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development.All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition.We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors.We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity.Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. [Display omitted] [Display omitted] DisclosuresKoppikar:Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

Abstract 4501: Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL

Abstract B-cell malignancies are hematologic disorders for which new therapeutic agents and novel treatment strategies are evolving. B cell receptor (BCR) signaling plays an important pathogenic role in diffuse large b-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). There is rapid clinical development of inhibitors targeting BCR-associated kinases such as Spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), and phosphoinositide 3-kinase (PI3K)δ. Recent clinical studies using these agents have demonstrated effectiveness and low toxicity in CLL and DLBCL patients with relapsed/refractory disease. With a great response rate, there are still a group of patients that do not respond to these therapeutic agents. The goal of this study is to investigate potential resistance mechanism(s) of BCR pathway inhibitors based on signature-signaling profiles. 21 CLL and DLBCL cell lines with different molecular features were assessed in this study to establish relevant signaling features and their association with sensitivities to BCR signaling inhibitors. First, we evaluated response of cell lines to small molecule inhibitors targeting the BCR signaling pathways (PCI-32765, R406, CAL 101, and Dasatinib). 28% (6/21) of the cell lines were resistant to PCI-32765; we found that these cells lines were also non responsive to CAL 101 and Dasatinib. Then we used phosphoflow assays and phosphoprotein arrays to profile BCR signaling in both responsive and non-responsive cell lines. The BCR signaling activities were assessed at baseline, after stimulation with IgG-IgM, and following pretreatment with phosphatase inhibitors. Interrogating pathway activity in cell lines with pervanadate, a phosphatase inhibitor, was especially revealing. After treatment with pervanadate, we observed an increase in phosphorylation status of SYK, BTK, p38, and S6 in normal B cells. However, 24% of tumor cell lines do not exhibit phosphorylation of BCR related proteins, SYK and BTK, after pervanadate treatment. Moreover, 83% (5/6) of cell lines resistant to PCI 32765 (IC50 > 20μM) did not respond to pervanadate, whereas only 12.5% (1/8) sensitive cell lines (IC50 < 2.5uM) did not respond to pervanadate. The difference observed in these cell lines indicates tumor cell lines with an abnormal BCR signaling may not be dependent upon BCR signaling for survival. More studies are being conducted to confirm this observation in patient samples and to further explore the molecular causes of the abnormal BCR signaling. We also explore combination treatments of PCI-32765, CAL-101 and other target therapy agents, which may provide useful insights for the optimal therapeutic use of these BCR signaling inhibitors in CLL and DLBCL. Overall, our results suggest that profiling of signaling network activities represents a promising approach to molecularly classifying DLBCL and CLL subtypes for target therapies. Citation Format: Joyce O. Obidi, Patricia Burke, Laura Richman, Dirk Mendel, Haifeng Bao. Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL. [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 4501. doi:10.1158/1538-7445.AM2014-4501

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer

AimPoly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. MethodsWe tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. ResultsIn a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. ConclusionTaken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.

Glutamate receptor antagonists in the management of migraine.

Migraine is a neurovascular disorder that is associated with severe headache and neurologic symptoms. The pathogenesis of migraine is believed to involve trigeminovascular system activation with the primary dysfunction located in brainstem. Glutamate, the major excitatory neurotransmitter in the central nervous system, and its receptors have since long been suggested in migraine pathophysiology. Different preclinical studies have confirmed their potential role in migraine. Moreover, several glutamate receptor modulators have been studied in clinical studies, some with promising results. In this review, we will give an overview of what is known about the role of glutamate in the pathogenesis of migraine, which will be followed by an overview of available efficacy, safety and tolerability data for glutamate receptor inhibitors in clinical development for the treatment of migraine.

Targeting hedgehog signaling in myelofibrosis and other hematologic malignancies.

Treatment of myelofibrosis (MF), a BCR-ABL–negative myeloproliferative neoplasm, is challenging. The only current potentially curative option, allogeneic hematopoietic stem cell transplant, is recommended for few patients. The remaining patients are treated with palliative therapies to manage MF-related anemia and splenomegaly. Identification of a mutation in the Janus kinase 2 (JAK2) gene (JAK2 V617F) in more than half of all patients with MF has prompted the discovery and clinical development of inhibitors that target JAK2. Although treatment with JAK2 inhibitors has been shown to improve symptom response and quality of life in patients with MF, these drugs do not alter the underlying disease; therefore, novel therapies are needed. The hedgehog (Hh) signaling pathway has been shown to play a role in normal hematopoiesis and in the tumorigenesis of hematologic malignancies. Moreover, inhibitors of the Hh pathway have been shown to inhibit growth and self-renewal capacity in preclinical models of MF. In a mouse model of MF, combined inhibition of the Hh and JAK pathways reduced JAK2 mutant allele burden, reduced bone marrow fibrosis, and reduced white blood cell and platelet counts. Preliminary clinical data also suggest that inhibition of the Hh pathway, alone or in combination with JAK2 inhibition, may enable disease modification in patients with MF. Future studies, including one combining the Hh pathway inhibitor sonidegib and the JAK2 inhibitor ruxolitinib, are underway in patients with MF and will inform whether this combination approach can lead to true disease modification.