Praechompoo Pongtornpipat

Synergistic induction of apoptosis in high-risk DLBCL by BCL2 inhibition with ABT-199 combined with pharmacologic loss of MCL1

Better treatments are needed for patients with diffuse large B-cell lymphoma (DLBCL) at high risk of failing standard therapy. Avoiding apoptosis is a hallmark of cancer, and in DLBCL the redundantly functioning antiapoptotic proteins BCL2 and MCL1 are frequently expressed. Here we explore drugs that cause loss of MCL1, particularly the potent new cyclin-dependent kinase inhibitor dinaciclib, which knocks down MCL1 by inhibiting CDK9. Dinaciclib induces apoptosis in DLBCL cells but is completely overcome by increased activity of BCL2. We find that clinical samples have frequent co-expression of MCL1 and BCL2, suggesting that therapeutic strategies targeting only one will lead to treatment failures owing to activity of the other. The BH3 mimetic ABT-199 potently and specifically targets BCL2. Single-agent ABT-199 had modest antitumor activity against most DLBCL lines and resulted in compensatory upregulation of MCL1 expression. ABT-199 synergized strongly, however, when combined with dinaciclib and with other drugs affecting MCL1, including standard DLBCL chemotherapy drugs. We show potent antitumor activities of these combinations in xenografts and in a genetically accurate murine model of MYC-BCL2 double-hit lymphoma. In sum, we reveal a rational treatment paradigm to strip DLBCL of its protection from apoptosis and improve outcomes for high-risk patients.

Evidence Suggesting That Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors.

The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of certain cancers, including 2% to 7% of non-small cell lung cancers (NSCLC) and ∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK(+) NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications.

Control of translational activation by PIM kinase in activated B-cell diffuse large B-cell lymphoma confers sensitivity to inhibition by PIM447

The PIM family kinases promote growth and survival of tumor cells and are expressed in a wide variety of human cancers. Their potential as therapeutic targets, however, is complicated by overlapping activities with multiple other pathways and remains poorly defined in most clinical scenarios. Here we explore activity of the new pan-PIM inhibitor PIM447 in a variety of lymphoid-derived tumors. We find strong activity in cell lines derived from the activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). Sensitive lines show lost activation of the mTORC1 signaling complex and subsequent lost activation of cap-dependent protein translation. In addition, we characterize recurrent PIM1 protein-coding mutations found in DLBCL clinical samples and find most preserve the wild-type proteins ability to protect cells from apoptosis but do not bypass activity of PIM447. Pan-PIM inhibition therefore may have an important role to play in the therapy of selected ABC-DLBCL cases.

TKI sensitivity patterns of novel kinase-domain mutations suggest therapeutic opportunities for patients with resistant ALK+ tumors

The anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions.

Oncogene Overdose: Too Much of a Bad Thing for Oncogene-Addicted Cancer Cells

Acquired resistance to targeted inhibitors remains a major, and inevitable, obstacle in the treatment of oncogene-addicted cancers. Newer-generation inhibitors may help overcome resistance mutations, and inhibitor combinations can target parallel pathways, but durable benefit to patients remains elusive in most clinical scenarios. Now, recent studies suggest a third approach may be available in some cases—exploitation of oncogene overexpression that may arise to promote resistance. Here, we discuss the importance of maintaining oncogenic signaling at “just-right” levels in cells, with too much signaling, or oncogene overdose, being potentially as detrimental as too little. This is highlighted in particular by recent studies of mutant-BRAF in melanoma and the fusion kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in anaplastic large cell lymphoma. Oncogene overdose may be exploitable to prolong tumor control through intermittent dosing in some cases, and studies of acute lymphoid leukemias suggest that it may be specifically pharmacologically inducible.

Target-Based Screening Against eIF4A1 Reveals the Marine Natural Compound Elatol as a Novel Inhibitor of Translation Initiation with In Vivo Anti-Tumor Activity

Purpose: The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors. Experimental Design: We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site mutagenesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing in vivo and assessed activity against xenografted tumors. Results: We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 in vitro with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent in vitro than the well-studied eIF4A1 inhibitor silvestrol but is tolerated in vivo at approximately 100× relative dosing, leading to significant activity against lymphoma xenografts. Conclusions: Elatols identification as an eIF4A1 inhibitor with in vivo antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy. Clin Cancer Res; 24(17); 4256–70. ©2018 AACR.

Abstract 5451: Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Activated Anaplastic Lymphoma Kinase (ALK) is seen in several cancers, including 2-7% of non-small cell lung cancer and ∼ 70% of Anaplastic Large Cell Lymphoma (ALCL). The most common ALK fusion in ALCL is a translocation, t(2;5)(p23;35), placing the kinase domain of ALK before the constitutively active promoter of nucleophosmin (NPM). Activated ALK drives oncogenesis by turning on multiple proliferative pathways. The success of tyrosine kinase inhibitors (TKIs) in ALK+ lung cancer has prompted evaluation of their use in ALK+ ALCL as a therapeutic strategy for patients who fail combination chemotherapy. Clinical studies in lung cancer show resistance to these drugs limits progression-free survival. We aim to identify resistance mechanisms against approved TKIs, crizotinib and ceritinib, in ALK+ ALCL. We selected for resistance in 3 patient-derived cell lines through propagation in gradually increasing drug concentrations. We assessed resistant subclones with long-insert whole-genome sequencing, viability and proliferation assays, qPCR and western blots. Similar profiling was carried out upon drug withdrawal. We validated our findings in cytokine-dependent murine pro-B cells transformed with NPM-ALK during ceritinib incubation. We xenografted resistant clones to SCID mice and assessed tumour burden over time. Resistant clones showed viability stimulation by TKIs and when washed out of drug, these clones underwent apoptosis due to hyper-stimulation of ALK signalling. Genomic amplification of NPM-ALK was seen leading to an increased expression at the mRNA and protein levels. TKI-dependence therefore was co-selected with resistance, showing for the first time toxicity to cancer cells due to an overdose of ALK signalling. We validated our findings in IL3-dependent FL/5.12 cells, which also showed co-selection for drug resistance and dependence due to ALK over-expression. Resistant ALCL lines selected for ability to grow without TKI behaved similarly to parent lines, showing no stimulation of viability in presence of drug. ALK mRNA and protein also return to baseline in these cells. SCID mice injected with resistant cells required TKI treatment for tumour engraftment and tumour burden decreased when drug dosing was stopped. When tumours re-grew, drug treatment was reinitiated, generating a second response demonstrating in vivo that NPM-ALK up-regulation results in both resistance and dependence on the TKI and resistant cells die from an overdose of ALK signalling in the absence of drug. Our results reveal that up-regulation of the fusion-ALK drug target is a resistance mechanism, previously un-reported in any ALK+ cancers. Up-regulation of NPM-ALK provides cells means to acquire resistance but also results in ALK overdose when drug is withdrawn, revealing intermittent dosing as a potential therapeutic strategy to prolong tumour control in ALK + patients. Citation Format: Soumya Sundara Rajan, Amit Dipak Amin, Matthew Groysman, Praechompoo Pongtornpipat, Jonathan Schatz. Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing. [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 5451. doi:10.1158/1538-7445.AM2015-5451