Kristen McEachern

The JAK2 Inhibitor AZD1480 Potently Blocks Stat3 Signaling and Oncogenesis in Solid Tumors

Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.

AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia

Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.

The Pim-1 Protein Kinase Is an Important Regulator of MET Receptor Tyrosine Kinase Levels and Signaling

ABSTRACT MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.

Novel Neutralizing Hedgehog Antibody MEDI-5304 Exhibits Antitumor Activity by Inhibiting Paracrine Hedgehog Signaling

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH. Mol Cancer Ther; 13(2); 386–98. ©2013 AACR.

Abstract 2796: Efficacy and biomarker modulation by AZD1208, a novel, potent and selective pan-Pim kinase inhibitor, in models of acute myeloid leukemia

The Pim serine/threonine kinase family is composed of three highly homologous members; Pim-1, Pim-2 and Pim-3, identified by the ability of the prototype member Pim-1 to drive lymphomagenesis in mice. Upregulation of Pim-1 and Pim-2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Overexpression of Pim-1 or Pim-3 has also been observed in prostate, pancreatic, gastric, bladder and hepatocellular cancers. Pim kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. Pims can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the activity of AZD1208, an orally available, potent and highly selective Pim inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of Pim-1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in phosphorylation of BAD, 4EBP1 and p70S6K. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of Pim targets. These results underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML. They also further support investigation of this inhibitor in other hematological and solid tumor malignancies where PIM signaling may play a role in tumorigenesis and survival. 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 2796. doi:1538-7445.AM2012-2796

Abstract CT147: Phase I studies of AZD1208, a PIM kinase inhibitor, in patients with recurrent or refractory acute myelogenous leukemia or advanced solid tumors

AZD1208 is a potent oral ATP-competitive, pan-PIM kinase inhibitor. Here we report the results of 2 Phase 1, open-label, multi-center dose escalation studies, recruiting patients with recurrent or refractory AML or advanced solid tumors including malignant lymphoma. The studies examined the safety, tolerability, pharmacokinetics and preliminary efficacy of AZD1208 in patients including evaluation of pharmacodynamic biomarkers in the AML study. The AML study was a first-in-patient study where 32 patients were treated and the range of treatment duration ranged from 15 to 27 days. In patients with heavily treated AML, AZD1208 was generally well tolerated up to doses of 700 mg QD, but was not tolerated at the 900 mg dose. The most common AEs reported likely to be associated with AZD1208 were nausea (37.5%), diarrhea (21.9%), vomiting (18.8%) and fatigue (18.8%). Three DLTs were reported including 1 case of Guillain-Barre syndrome at the 700mg dose and 2 cases of rash at the 900mg dose. Although there were no AML responses to AZD1208 treatment, reductions in peripheral blasts were seen in 5 patients. Modulation of PIM mediated biomarkers was observed with a greater than 50% reduction in pBAD in 14 out of 20 patients and a reduction in p4EBP1 in 6 out of 14 patients. There was no correlation of biomarker modulation and peripheral blast reduction. The solid tumor study recruited 35 patients with a median treatment duration of 41 days (range of 10-357 days). The best objective response was stable disease. Similar AEs to those seen in the AML study were observed in the solid tumor study. DLTs were reported in 4 patients including 2 patients with fatigue (800mg), increased GGT (240mg) and vomiting (540mg). In this study, AZD1208 was well tolerated as monotherapy at doses up to 700 mg QD, but was not tolerated at 800 mg QD. The MTD of AZD1208 was not determined in either study due to termination of development, however was below 900 mg in the AML study and below 800 mg in the solid tumor study. The PK of AZD1208 in both studies showed similar characteristics and was highly variable and generally dose proportional across the range of 120 mg to 900 mg. In the solid tumor study the absorption following multiple dosing was rapid, exposure showed time-dependent PK and interestingly, the accumulation ratios of exposure decreased with increasing doses. One possible mechanism for the decreased exposure is through increased CYP3A4 activity, demonstrated from a 4β-hydroxycholesterol assay showing that AZD1208 increased CYP3A4 activity after continuous dosing resulting in increased clearance of AZD1208 and perhaps resulting in decreased clinical activity. Overall, AZD1208 was generally well tolerated; however, there was no clear evidence of anti-tumor activity from AZD1208 monotherapy treatment. Citation Format: Jorge Cortes, Kenji Tamura, Daniel J. DeAngelo, Johann de Bono, David Lorente, Mark Minden, Geoffrey L. Uy, Hagop Kantarjian, Karen Keating, Kristen McEachern, Karthick Vishwanathan, Robert E. Godin, Janet Elizabeth Pease, Emma Dean. Phase I studies of AZD1208, a PIM kinase inhibitor, in patients with recurrent or refractory acute myelogenous leukemia or advanced solid tumors. [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 CT147.

Phase I studies of AZD1208, a proviral integration Moloney virus kinase inhibitor in solid and haematological cancers

BackgroundProviral integration Moloney virus (PIM) kinases (PIM1, 2 and 3) are overexpressed in several tumour types and contribute to oncogenesis. AZD1208 is a potent ATP-competitive PIM kinase inhibitor investigated in patients with recurrent or refractory acute myeloid leukaemia (AML) or advanced solid tumours.MethodsTwo dose-escalation studies were performed to evaluate the safety and tolerability, and to define the maximum tolerated dose (MTD), of AZD1208 in AML and solid tumours. Secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics (PD) and preliminary efficacy of AZD1208.ResultsSixty-seven patients received treatment: 32 in the AML study over a 120–900 mg dose range, and 25 in the solid tumour study over a 120–800 mg dose range. Nearly all patients (98.5%) in both studies experienced adverse events, mostly gastrointestinal (92.5%). Dose-limiting toxicities included rash, fatigue and vomiting. AZD1208 was not tolerated at 900 mg, and the protocol-defined MTD was not confirmed. AZD1208 increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance. There were no clinical responses; PD analysis showed biological activity of AZD1208.ConclusionsDespite the lack of single-agent clinical efficacy with AZD1208, PIM kinase inhibition may hold potential as an anticancer treatment, perhaps in combination with other agents.

Abstract 651: Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors

While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, including the overall presence and localization of immune cells, as well as the up-regulation of additional T-cell checkpoints, including TIM-3 in tumor infiltrating lymphocytes. To investigate the potential role of TIM-3, we set out to evaluate the expression and function of TIM-3 and the relationship to PD-1 in several systems. Firstly, we developed a flow cytometry methodology to enumerate T-cell, including CD4 and CD8 positive cells, as well as other immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, we investigated the expression profiles of TIM-3 and PD-1 in both T-cell and non-T-cell populations. In addition, we performed complementary genomic studies to explore gene expression profiles of not only the bulk tumor samples but also of isolated PD1 positive/TIM-3 negative versus PD1 and TIM-3 double positive cell populations. The profiling identified tumors with a range of tumor infiltrating lymphocyte content and also differences in PD1 and TIM-3 expression. It was found that in addition to T-cells, TIM-3 was expressed on myeloid cell populations and furthermore, that there were distinct differences in the gene expression profiles of PD1 single positive versus PD1 and TIM-3 double positive cells. Building on the expression studies, the functional role of Tim-3 in T-cells and also on myeloid-derived cells was explored. Taken together, these studies provide further evidence for TIM-3 as an important immunological checkpoint and as a relevant therapeutic target for the treatment of cancer with the potential for biological effects in both the T-cell and myeloid compartments of the tumor microenvironment. Citation Format: Kristen McEachern, Srimoyee Ghosh, Yonghong Zhao, Qiyao Zhang, Norman Zhang, David W. Jenkins. Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors [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 651. doi:10.1158/1538-7445.AM2017-651

Abstract 5600: Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC)

Introduction: The ineffective anti-tumor immune response is characterized by increased immune suppressive signals in the tumor microenvironment. In particular, T-cells recognizing tumor antigens can express diverse immune inhibitory receptors mediating lymphocyte inactivation and limiting tumor rejection. Blockade of these receptors such as PD-1 induces prominent clinical benefit in patients with NSCLC. However, the expression and significance of additional potentially actionable immune inhibitory receptors in lung cancer is poorly understood. Methods: After careful validation of assays and using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (clone EH33, CST), LAG-3 (Clone 17B4, Abcam) and TIM-3 (clone D5D5R, CST) in 698 stages I-IV formalin-fixed paraffin embedded (FFPE) lung carcinomas represented in three tissue microarrays (cohort #1 [Yale n=186], cohort #2 [Yale n=192, and cohort #3 [Greece n=320]). We also included a collection of lung adenocarcinomas with molecular annotation (cohort #4 [Yale n=106]). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers and with major clinico-pathological variables, driver mutations and survival were studied. Results: All the targets were detected predominantly in CD3+ T-cells with membranous staining. Expression of PD-1, LAG-3 and TIM-3 in T-cells across all NSCLC cohorts was 68.7%, 39.7% and 55.8%, respectively. Elevated levels of PD-1, LAG-3 or TIM-3 were significantly associated with increased tumor infiltrating lymphocytes and with the co-expression of one or more of the other inhibitory receptors (P Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in NSCLC, show frequent co-expression and association with elevated CD3+ T-cells. Our results support the biological role of PD-1, LAG-3 and TIM-3 in NSCLC and suggest co-activation of these immune inhibitory pathways in a proportion of cases. Modulation of these receptors could enhance the anti-tumor immune response in lung cancer. Citation Format: Ila J. Datar, Jun Wang, Nikita Mani, Franz Villarroel-Espindola, Patrick Ryan, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, David L. Rimm, Leiping Chen, Roy Herbst, Kurt Schalper. Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC) [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 5600. doi:10.1158/1538-7445.AM2017-5600

Abstract 3558: Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis. Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel. These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value. These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma. Citation Format: Kristen McEachern, Greg O’Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma. [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 3558.