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Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

Key Points CRISPR/Cas9 genomic editing of wild-type hematopoietic stem cells generates Npm1-Alk, leading to ALK+ large-cell lymphomas in recipients. CD30+ postthymic T-cell lymphomas are polyclonal but transplantable to secondary recipients with long latency.

// Amit Dipak Amin 1, * , Lingxiao Li 1, * , Soumya S. Rajan 2 , Vijay Gokhale 3, 4 , Matthew J. Groysman 5 , Praechompoo Pongtornpipat 3 , Edgar O. Tapia 6 , Mengdie Wang 6 , Jonathan H. Schatz 1 1 Department of Medicine, Division of Hematology-Oncology, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA 2 Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, FL, USA 3 BIO5 Institute, University of Arizona, Tucson, AZ, USA 4 Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA 5 Undergraduate Biology Research Program, University of Arizona, Tucson, AZ, USA 6 Cancer Biology Graduate Interdisciplinary Program, University of Arizona, Tucson, AZ, USA * These authors contributed equally to this work Correspondence to: Jonathan H. Schatz, e-mail: jschatz@miami.edu Keywords: anaplastic lymphoma kinase, drug resistance, crizotinib, ceritinib, alectinib Received: May 22, 2015 Accepted: March 02, 2016 Published: March 18, 2016 ABSTRACT 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.

Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies. Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3' ALK kinase domain to the 5' oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients. Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients. We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. Disclosures No relevant conflicts of interest to declare.

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. Cancer Res; 75(14); 2916–27. ©2015 AACR.

Anaplastic Large Cell Lymphoma (ALCL) is a rare T-cell lymphoma. The Anaplastic Lymphoma Kinase (ALK)-positive molecular subtype of ALCL is driven by constitutive activation of the ALK tyrosine-kin...

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