Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

Pim kinases are upregulated in leukemias and lymphomas and mediate cell growth and survival. Acute myeloid leukemia cell lines sensitive to the pan-Pim kinase inhibitor AZD1208 showed elevated pSTAT5 and Pim-1 expression that correlated with known tyrosine kinase mutations. However, one of the most sensitive lines, MOLM-16, lacked a reported driver mutation. RNA transcriptome sequencing identified a novel fusion gene comprised of the TYK2 kinase domain fused to the N-terminus of the RNA binding protein ELAVL1/HuR. The genes are co-localized within ∼2 MB on the short arm of chromosome 19, at position 13.2. The fusion results in loss of the Tyk2 pseudokinase domain, which negatively regulates kinase activity, suggesting a gain of function genetic alteration. Tyk2 was highly expressed and found to be amplified in MOLM-16 cells, with a higher degree of amplification of the exons encoding the kinase domain. Knockdown experiments demonstrated that down-regulation of the fusion gene, but not of the wild type ELAVL1 or TYK2 transcripts, resulted in suppression of STAT3/5 phosphorylation, Pim1 levels, and proliferation. Conversely, expression of the ELAVL1-TYK2 fusion protein in FDCP1 cells was shown to confer IL-3 independent growth. The fusion was not identified in screening of ∼200 AML patient samples or through analysis of TCGA data suggesting that it occurs at a low frequency, but interestingly NPM1-Tyk2 fusions have recently been described in cutaneous CD30-positive lymphoproliferative disorders (Velusamy et al. Blood 2014). Citation Format: Kristen McEachern, Erika Keeton, Keith Dillman, Minwei Ye, Chloe Stengel, Huawei Chen, Suping Wang, Shaun Grosskurth, Rosemary E. Gale, David C. Linch, Asim Khwaja, Zhongwu Lai, Dennis Huszar. A novel ELAVL1-TYK2 fusion protein drives STAT3/5 activation and PIM-1 expression, survival and growth in the MOLM-16 acute myeloid leukemia cell line. [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 2063. doi:10.1158/1538-7445.AM2015-2063

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-2antagonistofcelldeath,4EBP1,p70S6K,andS6,aswellasincreasesincleavedcaspase3 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. ( Blood . 2014;123(6):905-913)

Diffuse large B cell lymphoma is generally treated by chemotherapy and there is an unmet medical need for novel targeted therapies or combination therapies. Using in vitro screening, we have identified the combination of ibrutinib, an inhibitor of the tyrosine kinase BTK, and AZD2014, an mTOR catalytic inhibitor, as being highly synergistic in killing ABC-subtype DLBCL cell lines. Simultaneous inhibition of BTK and mTOR causes apoptosis both in vitro and in vivo and results in tumor regression in a xenograft model. We identify two parallel mechanisms that underlie apoptosis in this setting: cooperative inhibition of cap-dependent translation, and the inhibition of an NF-κB/IL10/STAT3 autocrine loop. Combined disruption of these pathways is required for apoptosis. These data represent a rational basis for the dual inhibition of BTK and mTOR as a potential treatment for ABC-subtype DLBCL.

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

Abstract Despite the success of Crizotinib in treating NSCLC patients with EML4-ALK fusions, cancers eventually develop resistance via a variety of mechanisms including mutations in the ATP binding site of the kinase domain. A series of 5-aminopyrazol-imidazopyridine compounds were identified to potently inhibit anaplastic lymphoma kinase and were found to be active against a number of mutations in vitro but suffered from strong Cyp inhibition and were found to be metabolically unstable by incubation in human hepatocytes and microsomes. Installation of a pyrimary alcohol group on the heteroaryl ethyl group of the scaffold consistently improved Cyp3A4 liability, and led to improvement in physical and DMPK properties as well as improvement of their metabolic stability in human Heps. The lead compound from this series was orally administrated to SCID mice in a Del xenograft model and achieved greater than 90% phospho-ALK inhibition over 6 hours post dose at 10 mg/kg dose. The lead compound was subsequently tested against Crizotinib-resistant ALK mutations in enzyme assays, and was shown to inhibit the clinically relevant ALK mutations, including L1196M. In enzyme assays, it was inactive against G1269S mutation, consistent with modeling studies. Subsequent profiling in an FDCP cell line over expressing ALK G1269S mutation showed potent anti-proliferative activity, suggesting off-target activity. Further characterization of this series identified AurB inhibition to be a key cell cycle kinase responsible for the off-target activity in the engineered cell line. The off target activity hinders it from assessing the primary pharmacology responsible for its preclinical efficacy in disease relevant models. In conclusion, we have identified a novel orally bioavailable compound that is a potent dual active ALK and AurB inhibitor that is active against clinically-relevant gatekeeper mutant. 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 3910. doi:1538-7445.AM2012-3910

Abstract 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

Cancerous cells emerge within the body following accumulation of deleterious genetic mutations. These mutations alter the phenotype of a cancer cell marking it as distinct from the surrounding host; an immunological state termed “altered self”. These cells, like other non-self entities such as viruses and bacteria, are recognised by the immune system and marked for destruction, a process known as “immune surveillance”. B7-H1 expression by tumour cells is believed to aid tumours in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumour infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumour micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumour cells from lysis by tumour cell specific cytotoxic T cells. Using hybridoma technology and high throughput screening MedImmune has identified a series of fully human antibodies specific for human B7-H1. Further characterisation of these antibodies led to the identification of a single high affinity antibody, MEDI 4736, with the ability to relieve B7-H1 mediated suppression of T cell activation in vitro and to enhance sub-optimal T cell activation in a mixed lymphocyte reaction. In vitro testing shows that MEDI 4736 does not trigger non-specific cytokine release in whole blood, and is only able to activate T cells in the context of an active T cell receptor signal. A surrogate anti-mouse B7-H1 antibody shows significant anti-tumour activity in a syngeneic model when dosed in combination with chemotherapy. Similarly MEDI 4736 is able to inhibit tumour growth in a novel in vivo xenograft model, via a mechanism that is dependent on the presence of tumour specific human T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2011-LB-158