Minhui Shen

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.

Discovery and Optimization of a Novel Series of Potent Mutant B-Raf V600E Selective Kinase Inhibitors.

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.

Identification of amidoheteroaryls as potent inhibitors of mutant (V600E) B-Raf kinase with in vivo activity.

A series of amidoheteroaryl compounds were designed and synthesized as inhibitors of B-Raf kinase. Several compounds from the series show excellent potency in biochemical, phenotypic and mode of action cellular assays. Potent examples from the series have also demonstrated good plasma exposure following an oral dose in rodents and activity against the Ras-Raf pathway in tumor bearing mice.

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B-Cell Lymphoma

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

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 919: AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib.

Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 60-80% anaplastic large cell lymphoma (ALCL) and 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR, cKIT and, more recently, IGF1R. We have discovered a novel and potent inhibitor of ALK, AZD3463 with a Ki value of 0.75nM which also inhibits additional receptor tyrosine kinases including insulin growth factor receptor (IGF1R) with equivalent potency. AZD3463 inhibits ALK in cells as demonstrated by its ability to decrease ALK autophosphorylation in tumor cell lines containing ALK fusions including DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK). Inhibition of ALK is associated with perturbations in downstream signaling including ERK, AKT and STAT3 pathways leading to preferential inhibition of proliferation in the ALK fusion containing cell lines in vitro. AZD3463 also demonstrates the ability to dose dependently inhibit pALK in xenograft tumors in vivo resulting in stasis (H3122) or regression (DEL, H2228). AZD3463 retains good activity against a number of clinically relevant crizotinib resistant mutations including the gatekeeper mutant L1196M where equivalent potency to wild type ALK is observed in vitro and in vivo in EML4-ALK containing BAF3 cell lines. To further assess the potential ability of AZD3463 to overcome additional resistance mechanisms, antiproliferative activity was assessed in multiple crizotinib resistant cell lines independently derived in vitro from H3122 cells as well as a patient derived crizotinib relapsed model. These resistant cell lines contain multiple resistance mechanisms including the L1196M gatekeeper and T115Ins mutations, ALK amplification and/or secondary drivers including EGFR and IGF1R. AZD3463 retains antiproliferative potency within 4 fold of parental H3122 cells for 10 out of 12 of these acquired resistance models in vitro. In summary, AZD3463 is a potent ALK inhibitor which inhibits additional kinases including IGF1R and has activity in a number of crizotinib resistant models driven by multiple resistance mechanisms. Citation Format: Lisa Drew, Jane Cheng, Jeffrey Engelman, Douglas Ferguson, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Alice Shaw, Minhui Shen, Dan Widzowski, Allan Wu, Graeme Smith. AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 919. doi:10.1158/1538-7445.AM2013-919

AZ304, a novel dual BRAF inhibitor, exerts anti-tumour effects in colorectal cancer independently of BRAF genetic status

BackgroundBRAF mutation is associated with poor clinical outcome of patients with malignant tumours, and mediates resistance to chemotherapy and targeted therapy. This study aimed to determine whether V600E mutant and wild type BRAF colorectal cancers exhibit distinct sensitivities to the dual BRAF inhibitor AZ304.MethodsKinase activity was assessed by the AlphaScreen assay. Then, MTT assay, EdU assay, colony-formation assay and Western blot were performed to evaluate the anti-tumour effects of AZ304 in vitro. In vivo efficacy was investigated by xenograft analysis and immunohistochemistry.ResultsAZ304 exerted potent inhibitory effects on both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF, with IC50 values of 79 nM and 38 nM, respectively. By suppressing ERK phosphorylation, AZ304 effectively inhibited a panel of human cancer cell lines with different BRAF and RAS genetic statuses. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. In addition, the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independently of BRAF mutational status.ConclusionsThe BRAF inhibitor AZ304 has broad spectrum antitumour activity, which is significantly enhanced by combination with Cetuximab in colorectal cancers in vitro and in vivo.

Abstract 3572: AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models

Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 Citation Format: Justin Cidado, Minhui Shen, Michael Grondine, Scott Boiko, Haiyun Wang, Alexandra Borodovsky, Anne Marie Mazzola, Alan Wu, Deborah Lawson, Douglas Ferguson, Beirong Gao, Andy Cui, Celina D’Cruz, Lisa Drew. AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models. [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 3572.

Abstract 3362: Miniaturized PBPK model improves pharmacodynamic characterization and physiological interpretability for compounds with profound hysteresis in tumor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Significant hysteresis between plasma concentration and target inhibition at the effect site (e.g., tumor) is a frequent observation, commonly described mathematically by connecting the central (i.e., plasma) compartment to an ‘effect compartment’ by a ‘link’ which causes the concentration in the latter to be delayed relative to the plasma. The result is a direct response between effect-compartment concentration and target inhibition. A significant drawback is that the effect compartment cannot be observed (making it impossible to validate) and has no physiological interpretation (rendering communication with other disciplines difficult). We develop a novel approach that is more physiologically meaningful, provides more-precise model parameter estimates, and gives insight into the physico-chemical factors limiting distribution into the tumor. Method: We orally administered single doses of several compounds (including Crizotinib, AZD3463, and others) targeting ALK to mice bearing tumors derived from the DEL and H3122 non-small-cell lung cancer line, at several dose levels. At 6, 24, and 48 hours post-dose, we measured the plasma and tumor concentrations of each compound and associated target inhibition (phosphorylated ALK, pALK) in the tumor. pALK inhibition shows a direct response not to plasma, but to tumor concentration, indicating that the delay is distributional in nature. We constructed a miniature physiologically-based pharmacokinetic (mPBPK) model consisting of a central compartment and a tumor of fixed physiological volume. pALK inhibition was modeled as a direct Emax response to tumor concentration. For each compound, we simultaneously fitted the mPBPK model to the naive-pooled plasma and tumor concentrations, as well as pALK, using all available dose levels. Beyond the standard PK and PD parameters (Emax, E, IC50) we also fitted the tumor partition constant Kp, and tumor blood flow rate Qt. For comparison, we fitted a standard effect-compartment (‘link’) model to the plasma concentrations and pALK levels to the same data. Results: For each compound, we computed unbound EC50 for both effect-compartment and mPBPK models. We found that while the point estimates largely agree, the mPBPK model delivers more-precise estimates (typically 50% lower CV%). We attribute this to its use of additional data (tumor concentration) to constrain the model, which more than compensates for the additional parameters in the mPBPK model. We find that there is broad consistency in estimates of tumor flow rate Qt across the compounds studied, indicating that distribution from plasma to site of action is limited by blood flow, rather than by permeability. Additionally, we found that the greater physiological interpretability of the mPBPK model enhances cross-functional communication within project teams. Citation Format: Francis D. Gibbons, Dan Widzowski, Minhui Shen, Jane Cheng, Lisa Drew, Jamal C. Saeh, Douglas Ferguson. Miniaturized PBPK model improves pharmacodynamic characterization and physiological interpretability for compounds with profound hysteresis in tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2013-3362

Abstract 4465: Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa.

Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR and cKIT. To further evaluate potential mechanisms of acquired resistance to crizotinib and potential therapeutic strategies to overcome them, we have developed multiple independently generated cell line derivatives. H3122 cells, a NSCLC line with an EML4-ALK fusion, was selected in increasing concentrations of crizotinib up to 1uM over a period of 4 to 6 months to generate H3122-CR lines. The cell lines generated were confirmed to be resistance to crizotinib as well as the selective ALK inhibitor, CH5424802, in cell proliferation experiments. Evaluation of the levels of potential secondary receptor tyrosine kinase (RTK) drivers by phosphoRTK arrays and western blotting revealed increased levels of total and phosphorylated ALK (pALK), along with increased phosphorylated insulin-like growth factor 1 receptor (pIGF1R). ALK levels were also elevated at the mRNA level. To evaluate potential strategies to most effectively overcome resistance in these cell lines, selective inhibitors of ALK CH5424802, IGF1R OSI-906 and epidermal growth factor receptor (EGFR) Iressa were used alone and in combination to evaluate ability to inhibit growth of two of the H3122-CR lines. In both CR lines, OSI-906 and Iressa had no effect alone on cell growth, however a combination of CH5424802 and OSI-906 or CH5424802 and Iressa increased sensitivity to CH5424802. Most interestingly however, a combination of CH5424802, OSI-906 and Iressa had maximal sensitization effect with complete reversal of resistance observed. A similar finding was obtained with a combination of the ALK/IGF1R inhibitor AZD3463 with Iressa. Further, we were able to demonstrate pathway dependency shifts in the CR lines with OSI-906 inhibiting AKT and Iressa inhibiting ERK and a combination of CH5424802, OSI-906 and Iressa required to effectively repress these main pathways to the level observed with CH5424802 alone in the H3122 parental line. Together, these data support a potential role for both EGFR and IGF1R as secondary RTK drivers occurring concurrently to drive resistance to crizotinib. A combination of approaches inhibiting ALK, IGF1R and EGFR together may therefore be required to most effectively treat crizotinib relapsed patients and delay onset of resistance. This could be achieved by combining multiple selective agents or agents with polypharmacology such as AZD3463 along with Iressa. Citation Format: Lisa Drew, Jeffrey Engelman, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Meghan Scarpitti, Alice Shaw, Minhui Shen, Suping Wang, Graeme Smith. Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2013-4465