Search

Supplementary Tables S1-S2. Relative IC50's of ALK+ and ALK- ALCL cell lines to the ALK tyrosine kinase inhibitors crizotinib and ceritinib (S1); ALK Kinase-Domain Sequencing Results (S2).

Supplementary Figures S1-S5. Timelines for acquired resistance and resensitization (S1); Assessment of copy number changes in additional subclones (S2); RS lines behave like original parent cells with return of ALK expression to baseline (S3); Reselection of resensitized cells again selects for up-regulation of NPM-ALK and the resistant-dependent phenotype (S4); Assessing whether an up-front intermittent dosing regimen prolongs tumor control in vivo (S5).

Supplementary References

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.

Supplementary Figure Legend. Legend for Supplementary Figures S1-S5.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial12, we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases13, we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1β and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand–receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development. Lung samples collected soon after death from COVID-19 are used to provide a single-cell atlas of SARS-CoV-2 infection and the ensuing molecular changes.

Non-small cell lung cancer (NSCLC) accounts for nearly half of all newly diagnosed patients with brain metastasis (BM), followed by melanoma and breast carcinomas. The presence of BM is associated with reduced response to several modern cancer therapies and a poor prognosis, but the underlying molecular underpinnings remain poorly understood. Here, we performed multi-modal single-nucleus RNA, T cell receptor, single-cell spatial, and whole-genome sequencing (WGS) of 44 primary NSCLC tumors (PTs) and BMs. Through combination of WGS with inferred copy-number alterations (CNAs) and gene expression snRNA-seq, we robustly identify malignant cells despite the presence of healthy cell mosaicism. We find a strong association of chromosomal instability (CIN) and brain-metastatic organotropism. Through integration with clinical information and thousands of publicly available whole-exome sequencing (WES) profiles obtained from patients with NSCLC, we validate this observation and show that CIN progressively increases from PTs to extracranial metastases (ECMs) and is the highest in BMs. Using non-negative matrix factorization, we identify recurrent transcriptional hallmarks cancer metastasis, and additionally find that cancer cells from BMs strongly enrich for a neuronal-like cell state. At single-cell resolution, we indeed identify a rare cancer cell population genomically define by very high CIN, and transcriptionally characterized by a program of epithelial-to-mesenchymal transition (EMT), neuronal-like differentiation, and loss of lineage attribution. We show in our data and external scRNA-seq data that this cell state does not ecist in healthy lungs, progressively enriches from PTs to ECMs, and is most abundant in BMs, suggesting that these cells may indeed give rise to BMs. Furthermore, through integration of snRNA/TCR-seq and spatial transcriptomics, we find distinct tumor-microenvironments across disease sites, including, nearly exclusive expansion of tissue-resident myeloid cells in PTs, while BMs are largely dominated by dense infiltration with monocyte-derived macrophages and granulocytes, impaired T cell infiltration, activation and clonal expansion. Lastly, spatial transcriptomics also recurrent, cell-type specific patterns of geographic variability in key pathways, including antigen presentation, EMT, oxidative phosphorylation, and inflammatory response and associated cellular micro-niches. Together, this work identifies cellular, genomic, and transcriptional features of NSCLC BMs and has important therapeutic implications for novel therapies, in particular immunomodulatory approaches targeting cell types/states unique to disease sites. Citation Format: Somnath Tagore, Lindsay Caprio, Amit Dipak Amin, Irving Barrera, Johannes Melms, Karan Luthria, Yiping Wang, Yohanna Georgis, Abhi Jaiswal, Galina G. Lagos, Zachary Walsh, Parin Shah, Jana Biermann, Neha Sheikh, Priyanka Ramaradj, Niroshana Anandasabapathy, Hanina Hibshoosh, Gary Schwartz, Brian Henick, Alison Taylor, Fei Chen, Benjamin Izar. Multi-modal single-cell and spatial genomics reveals genomic, adaptive and microenvironmental features of human non-small cell lung cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3517.

Non-small cell lung cancers (NSCLCs) harboring deletions or inactivating mutations in STK11 (encoding LKB1) are associated with treatment-resistance, including to immune checkpoint blockade, and poor survival, yet the underlying mechanisms are poorly understood. Here, we combined multi-modal single-cell transcriptomics, whole-genome sequencing (WGS), and analysis of public data bases totaling >10,000 NSCLC whole-exome sequencing (WES) and when available RNA (RNA-seq) profiles of AACR GENIE, MSK IMPACT, TCGA and CPTAC, high-content imaging, and functional assays, to determine genomic and mechanistic features of STK11-mutant NSCLC. Across human WES/RNA-seq data, we find that STK11-mutant NSCLC have a significantly higher fraction of genome altered (FGA) and score strongly for the mRNA-based CIN70 signature compared to other NSCLC genotypes, overall indicating that these tumors have a higher degree of chromosomal instability (CIN). Using high-content imaging, we show that both human and murine STK11-mutant models have a higher rate of micronuclei and chromosome-mis-segregation events, confirming their CINhigh status in dynamic assays. Next, we show that tonic CIN-induced activation of cGAS-STING signaling, whose activation is typically thought of inducing type I interferon expression, results in strong suppression of anti-tumor immune signaling. Remarkably, genetic or pharmacologic suppression of cGAS, and thus depletion of the STING ligand cGAMP, results in reprogramming and re-sensitization of STK11-mutant models to cGAS-STING mediated type I interferon responses. Furthermore, we show that CIN promotes excessive production cGAMP in STK11-mutant models. We find upregulation of two ectonucleotidases (ENPP1 and CD73/NT5E) that hydrolyze exported cGAMP to adenosine, which may further contribute to the highly dysfunctional tumor-microenvironment observed in STK11-mutant tumors. Lastly, suppression of CIN through overexpression of mitotic-centromere associated kinesin (MCAK) in murine STK11-mutant models results in decreased tumor growth and sensitization to anti-PD1 therapy. In summary, we define STK11-mutant as archetypical chromosomally form of NSCLC, provide mechanistic basis that corelates with poor clinical outcomes, and demonstrate how relief of tonic CIN-induced changes may be therapeutically leveraged. Citation Format: Lindsay A. Caprio, Christy Hong, Amit Dipak Amin, Somnath Tagore, Johannes Melms, Luke Cai, Yiping Wang, Patricia Ho, Michael Mu, Hanina Hibshoosh, Brian Henick, Kwok K. Wong, Samuel F. Bakhoum, Benjamin Izar. Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5944.

The cell autonomous balance of immune-inhibitory and -stimulatory signals is a critical yet poorly understood process in cancer immune evasion. Using patient-derived co-culture models and humanized mouse models, we show that an intact CD58:CD2 interaction is necessary for anti-tumor immunity. Defects in this axis lead to multi-faceted immune evasion through impaired CD2-dependent T cell polyfunctionality, T cell exclusion, impaired intra-tumoral proliferation, and concurrent protein stabilization of PD-L1. We performed genome-scale CRISPR-Cas9 and CD58 coimmunoprecipitation mass spectrometry screens identifying CMTM6 as a key stabilizer of CD58, and show that CMTM6 is required for concurrent upregulation of PD-L1 in CD58 loss. Single-cell RNA-seq analysis of patient melanoma samples demonstrates that most TILs lack expression of primary costimulatory signals required for response to PD-1 blockade (e.g. CD28), but maintain strong CD2 expression, thus providing an opportunity to mobilize a so far therapeutically untapped pool of TILs for anti-tumor immunity. We identify two potential therapeutic avenues, including rescued activation of human CD2-expressing TILs using recombinant CD58 protein, and targeted disruption of PD-L1/CMTM6 interactions. Our work identifies an underappreciated yet critical axis at the nexus of cancer immunity and evasion, uncovers a fundamental mechanism of co-inhibitory and -stimulatory signal balancing, and provides new approaches to improving cancer immunotherapies.

Single-cell genomics are enabling technologies, but their broad clinical application remains challenging. We report an easily adaptable approach for single-cell transcriptome and T cell receptor (TCR)-sequencing, and matched whole-genome sequencing from tiny, frozen clinical specimens. We achieve similar quality and biological outputs while reducing artifactual signals compared to data from matched fresh tissue samples. Profiling sequentially collected melanoma samples from the KEYNOTE-001 trial, we resolve cellular, genomic, and clonotype dynamics that encapsulate molecular patterns of tumor evolution during anti-PD-1 therapy. To demonstrate applicability to banked biospecimens of rare diseases, we generate a large uveal melanoma liver metastasis single-cell and matched WGS atlas, which revealed niche-specific impairment of clonal T cell expansion. This study provides a foundational framework for propelling single-cell genomics to the clinical arena.

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.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma, yet our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naïve MBM and 10 extracranial melanoma metastases (ECM), and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion, and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX(+)CD8(+) T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Genomic and adaptive determinants of organ-specific metastasis are poorly understood. A model of sequential acquisition of divergent somatic mutations is insufficient to explain metastasis. Liver metastasis (LM) occurs frequently and is associated with a poor prognosis and reduced therapy response in several cancers, including in patients with melanoma and lung cancer. To identify drivers of metastatic niches, we used a syngeneic mouse melanoma model which recapitulates genomic, metastatic and therapy response patterns seen in patients. We performed a large-scale in vivo CRISPR-Cas9 knockout screen and identified perturbations that promote LM, but not primary tumor growth or metastasis to other organs (e.g. lung). The “top hit” in this screen associated with LM was loss Pip4k2c. We generated Pip4k2cKO cells and show that in otherwise isogenic melanoma cell lines, loss of Pip4k2c led to increased baseline and insulin-induced activation of the PI3K/AKT pathway, and increased invasive capacity. Rescuing Pip4k2cKO with full-length (Pip4k2cRec) or allosteric domain deficient (Pip4k2cAD) Pip4k2c ORFs, we show that hyperactivation of the PI3K/AKT pathway in is mediated by loss of the allosteric domain function, and not loss of the kinase domain of Pip4k2c. Treatment with different PI3K inhibitors effectively abrogated the pathway, but was partly bypassed in the presence of insulin in Pip4k2cKO and Pip4k2cAD, but not parental or Pip4k2cRec cells. Upon tail vein injection, Pip4k2cKO cells produced a significantly increased LM burden compared to parental cells, and this effect was rescued in Pip4k2cRec but not Pip4k2cAD, further affirming that loss of allosteric domain was required for this phenotype. We reasoned that Pip4k2cKO cells preferentially colonized the liver by co-opting the insulin-rich milieu in this organ. To test this, we used shRNA targeted against the insulin receptor (Insr) generated Pip4k2cKO/InsrshIR and showed that Insr was required but not sufficient to enhance LM burden. Given the promising in vitro activity of PI3K inhibitors, we next tested whether these could abrogate LM in vivo. Surprisingly, we found a substantial increase in LM burden in mice with Pip4k2cKO-bearing LM treated with PI3K inhibition compared to vehicle treated animals. We show that this paradoxical observation was due to host-mediated increased in glucose and insulin in response to PI3K inhibitor, which promoted a forward loop of increased liver metastasis. Breaking this loop with either ketogenic diet or treatment with a SGLT2 inhibitor in turn rescued increased these host responses and resulted in reduced LM burden in combination with PI3K inhibition. In summary, we identify a novel mechanism of metastatic liver organotropism and pharmacological and dietary combinations to reduced liver metastatic burden. Given the expanding use of PI3K inhibitors, our findings may have important clinical implications. Citation Format: Meri Rogava, Johannes C. Melms, Stephanie Davis, Clemens Hug, Bryan Ngo, Michael J. Lee, Patricia Ho, Amit Dipak Amin, Yiping Wang, Sean Chen, William Ge, David Liu, Thomas Tüting, Martin Röcken, Thomas K. Eigentler, Samuel F. Bakhoum, Andrei Molotkov, Akiva Mintz, Lewis C. Cantley, Peter K. Sorger, Benjamin Izar. A genetic-metabolic axis of metastatic liver organotropism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 981.

Brain metastases are the most frequent malignancies in the brain and are associated with significant morbidity and mortality. Melanoma brain metastases (MBM) occur in most patients with advanced melanoma and are challenging to treat. Our understanding of the treatment-naïve landscape of MBM is still rudimentary, and there are no site-specific molecular therapies available. To gain comprehensive insights into the niche-specific biology of MBM, we performed multi-modal profiling of fresh and frozen samples using single-cell RNA-seq, single-cell TCR-seq, single-nuclei RNA-seq, and spatial transcriptional profiling. We evolved single-nucleus RNA-seq processing methods to enable profiling of minute amounts of archival, frozen specimens and compared data quality and structure between matched fresh and frozen MBM. We curated a treatment-naïve single-transcriptome atlas of MBM, collected either fresh samples over 1 year or profiled frozen samples dating back more than 15 years, and compared these samples to extracranial melanoma metastases (ECMM). In total, we profiled 25 samples with more than 114,000 transcriptomes. We identified more than 20 different cell types, including diverse tumor-infiltrating T-cell subsets and rare dendritic cell types, and tissue-specific cell types, such as activated microglia. Tumor cells in MBM showed an increase in copy number alterations (CNAs) compared to ECMM, which we validated using an external dataset of whole exome sequencing (WES) data including both MBM and ECMM. MBM-derived tumor cells show enrichment of genes involved in neuronal development and function, and site-specific metabolic programs (e.g., oxidative phosphorylation). Comparison with an external bulk RNA-seq dataset validated enriched key genes in MBM and ECMM as putative dependencies. We recovered cell-cell interactions between tumor and brain-resident cells involved in brain development, homeostasis, and disease. Similar to ECMM, the tumor microenvironment of MBM contained CD8+ T cells across a spectrum of differentiation, exhaustion and expansion, which was associated with loss of TCF7 expression and adoption of a TOX+ cell state. CD4+ T cells included T regulatory, T helper and T follicular-helper-like expression profiles. Plasma cells showed spatially localized expansion and limited heterogeneity. Myeloid cells largely adopted pro-tumorigenic cell states, including microglia, the brain-resident myeloid cells, which showed an activation trajectory characterized by expression of SPP1 (osteopontin). Spatial transcriptional analysis revealed restricted expression of antigen presentation genes with only a subset of these locations showing a type I interferon response. In summary, this work presents a multi-modal single-cell approach to dissect and compare the landscape of treatment-naïve MBM and ECMM. Citation Format: Johannes C. Melms, Jana Biermann, Amit Dipak Amin, Yiping Wang, Somnath Tagore, Massimo Andreatta, Ajay Nair, Meri Rogava, Patricia Ho, Lindsay A. Caprio, Zachary H. Walsh, Shivem Shah, Daniel H. Vacarro, Blake Caldwell, Adrienne M. Luoma, Joseph Driver, Matthew Ingham, Suthee Rapisuwon, Jennifer Wargo, Craig L. Slinguff, Evan Z. Macosco, Fei Chen, Richard Carvajal, Michael B. Atkins, Michael A. Davies, Elham Azizi, Santiago J. Carmona, Hanina Hibshoosh, Peter D. Canoll, Jeffrey N. Bruce, Wenya L. Bi, Gary K. Schwartz, Benjamin Izar. Dissecting the ecosystem of treatment-naïve melanoma brain metastasis using multi-modal single-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 984.

e23518 Background: Single-cell RNA-seq is an enabling technology that may inform the molecular underpinnings of drug response and resistance in patient biopsies. This method is difficult to implement in the study of rare diseases such as sarcomas due to specimen requirements and technical limitations. Methods: Here, we evolved novel methods that we recently reported in melanoma ( Wang, Fan, et al., bioRxiv, 2022), which enable single-nucleus RNA, T cell receptor (snRNA/TCR)-seq, and pool-matched whole-genome sequencing (WGS) from archival, frozen sarcoma tissue. Results: This enabled profiling of 75,716 cells and 788 matched TCR clonotypes from six patients with intimal sarcoma (INS) and undifferentiated pleomorphic sarcomas (UPS), including two matched pair samples from pre/post-immune checkpoint inhibitor (ICI). Our analysis revealed substantial transcriptional cancer cell heterogeneity driven by varying copy number alterations (CNAs). In one patient with INS with a complete response to ICI followed by an isolated recurrence, we identified a rare cancer cell clone defined by CNA (confirmed with WGS) and resulting transcriptional outputs that pre-existed and emerged during resistance. Furthermore, in a patient with UPS with intrinsic resistance to ICI, we find adequate T cell clonal expansion and activation, suggesting appropriate T cell response to ICI dampened by intrinsic mechanisms of ICI resistance within the cancer cells. Non-negative matrix factorization (NMF) analysis identified cell states associated with either intrinsic or adaptive resistance to ICI that was distinct from resistance to doxorubicin. These observations are consistent with those previously reported from sequential biopsies obtained from KEYNOTE-001 in metastatic melanoma ( Wang, Fan, et al., bioRxiv, 2022), which also revealed emergence of pre-existing populations of resistant clones defined by their underlying aneuploidy patterns. Conclusions: Together, these results demonstrate feasibility of implementing single-cell genomics from archival tissue to study sarcoma and propel our understanding of drug resistance. Conceptually, this work suggests that large-scale CNAs may drive cell sub-populations associated with ICI resistance in sarcoma and in other diseases.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality, develops almost exclusively in patients with chronic liver disease (CLD) and advanced fibrosis(1,2). Here we interrogated functions of hepatic stellate cells (HSC), the main source of liver fibroblasts(3), during hepatocarcinogenesis. Genetic depletion, activation or inhibition established HSC as tumour-promoting in mouse models of HCC. HSC were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analysis of mouse and human HSC subpopulations and their associated mediators by single cell RNA-sequencing in conjunction with genetic ablation revealed dual functions of HSC in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSC (cyHSC), protected from hepatocyte death and HCC development. In contrast, type I collagen, enriched in activated myofibroblastic HSC (myHSC), promoted proliferation and tumour development via increased stiffness and TAZ activation in pretumoural hepatocytes and via activation of discoidin domain receptor 1 in established tumours. An increasing HSC dysbalance between cyHSC and myHSC during liver disease progression was associated with elevated HCC risk in patients. In summary, the dynamic shift of HSC subpopulations and their mediators during CLD is associated with a switch from HCC protection to HCC promotion.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection

A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased β-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (pin vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

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.

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.

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: Elatol's 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.

Gene-expression profiling and next-generation sequencing have defined diffuse large B-cell lymphoma (DLBCL), the most common lymphoma diagnosis, as a heterogeneous group of subentities. Despite ongoing explosions of data illuminating disparate pathogenic mechanisms, however, the five-drug chemoimmunotherapy combination R-CHOP remains the frontline standard treatment. This has not changed in 15 years, since the anti-CD20 monoclonal antibody rituximab was added to the CHOP backbone, which first entered use in the 1970s. At least a third of patients are not cured by R-CHOP, and relapsed or refractory DLBCL is fatal in ∼90%. Targeted small-molecule inhibitors against distinct molecular pathways activated in different subgroups of DLBCL have so far translated poorly into the clinic, justifying the ongoing reliance on R-CHOP and other long-established chemotherapy-driven combinations. New drugs and improved identification of biomarkers in real time, however, show potential to change the situation eventually, despite some recent setbacks. Here, we review established and putative molecular drivers of DLBCL identified through large-scale genomics, highlighting among other things the care that must be taken when differentiating drivers from passengers, which is influenced by the promiscuity of activation-induced cytidine deaminase. Furthermore, we discuss why, despite having so much genomic data available, it has been difficult to move toward personalized medicine for this umbrella disorder and some steps that may be taken to hasten the process.

The histone genes are an important group of cell cycle regulated genes whose transcription is activated during the G1/S transition and repressed in early G1, late S, and G2/M. The HIR complex, comprised of Hir1, Hir2, Hir3 and Hpc2, regulates three of the four histone gene loci. While relief of repression at the G1/S boundary involves the HIR complex, as well as other cofactors, the mechanism by which this derepression occurs remains unknown. To better understand how transcriptional repression contributes to periodic expression in the cell cycle, we sought to identify the cell cycle signals required to alleviate HIR-mediated repression of the histone genes. By measuring histone gene transcription in strains with various combinations of clb mutations, we found that the mitotic Clb1/Clb2 cyclins are required to alleviate Hir-mediated repression during the G1/S transition and that Clb2 physically interacts with the HIR complex. While the HIR complex regulates histone genes transcription in combination with two other histone H3/H4 chaperones, Asf1 and Rtt106, our data demonstrate that the mitotic Clb cyclins are necessary to specifically alleviate the repressive action of the HIR complex itself in order to allow proper expression of the histone genes in late G1/early S phase.

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae . Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae . We showed that the N terminal CDI domain (spanning amino acids 63–79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452–480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.

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.

Aberrant protein kinase activity promotes tumor survival and proliferation, and targeted kinase inhibitors that halt growth and promote apoptosis demonstrate some cancers are truly kinase addicted. Clinically, this is best exemplified by chronic myeloid leukemia (CML), driven by the fusion kinase BCR-ABL, where tyrosine kinase inhibitor (TKI) therapy can control the disease for years, perhaps indefinitely in many patients. For other cancers, however, the success of kinase inhibition has been more modest. Despite great strides in drug design and delivery, resistance invariably develops, typically limiting median progression free survival (PFS) to a period of months. Development of new-generation inhibitors therefore has focused on increasing potency, overcoming resistance-conferring mutations to the drug target, and hitting parallel signaling pathways that bypass the target altogether. While sequential treatments and/or combination cocktails to circumvent resistance may work in some cases, concerns arise regarding toxicity and cost, prompting exploration of innovative new strategies to prolong PFS. Two recent studies in different cancers propose an alternative with a potential to increase the duration of tumor control by several already approved TKIs. Approximately 70% anaplastic large cell lymphoma (ALCL) cases are driven by the constitutively activated fusion kinase NPM-ALK [1]. To investigate resistance mechanisms, our laboratory grew patient-derived NPMALK-driven cell lines in one of two FDA approved ALK TKIs, crizotinib or ceritinib, at increasing concentrations. Resistance reliably arose due to overexpression of NPM-ALK even if resistanceconferring mutations also began to arise. Strikingly, characterization of resistant phenotypes showed viability of these cells was actually stimulated by – indeed required – continued presence of ALK TKI, as drug withdrawal rapidly induced apoptosis. Concomitantly we observed massive ALK activation, suggesting over-activation provides as much of a fitness deficit as inhibition [2]. These results echo the findings of a prominent study investigating mutant-BRAF inhibition with vemurafenib in melanoma, where resistance also arose due to target overexpression [3]. As in our study, resistant cells underwent apoptosis in response to inhibitor withdrawal. Oncogene over-expression in both reports therefore promoted a dual phenotype of drug resistance and dependence. Both studies demonstrate potential therapeutic exploitation of the paradoxically toxic response of resistant-dependent cells to drug withdrawal. Xenografted resistant cells in both reports underwent apoptosis leading to tumor regressions upon discontinuation of drug dosing to host animals. After time tumors resumed growth, but sampling showed drug-target expression had returned to baseline – a requirement for their growth without inhibitor. At the same time dependence went away, so did resistance, as re-initiation of inhibitor dosing to host animals led to new rounds of tumor regressions. This suggested cycling of drug through discontinuous dosing could forestall onset of fatal resistance, and was explored in both reports, but especially in the melanoma models [2, 3]. Here both pre-scheduled and individualized intermittent dosing strategies greatly prolonged tumor control compared to continuous drug administration. Patients at risk of developing resistance due to upregulation of some oncogenes may therefore benefit from intermittent dosing, a strategy carrying both low cost and low toxicity. A randomized phase 2 trial comparing intermittent vs. continuous inhibitor dosing in melanoma already is enrolling patients ({"type":"clinical-trial","attrs":{"text":"NCT02196181","term_id":"NCT02196181"}}NCT02196181), and one is in planning for ALK+ ALCL. More preclinical assessment, however, also is needed in these and other cancers. Supporting the approach, drug holidays already may be employed to counter toxicity, and some second remissions to the ALK TKI crizotinib have been reported in lung cancer patients whose tumors were previously resistant [4, 5]. Great care must be taken, when determining appropriate timing of drug administration and withdrawal with such strategies, as the onset of resistance may be unpredictable, and drug interruption or re-initiation too early could exacerbate the onset of other resistance pathways [6]. An intriguing alternative, however, is identifying the specific mechanisms by which over-activity of particular oncogenes promote toxicity, and then pharmacologically inducing them as either a direct means of cellular toxicity or to prime the cells for other therapies. Indeed, a recent study showed pharmacological induction of SYK hyper-activation caused BCR-ABL+ acute lymphoblastic leukemia (ALL) cell death [7]. Targeted inhibition of several signaling pathway targets downstream failed to rescue resistant cells from NPM-ALK overdose in our systems (unpublished observations), but unbiased approaches are ongoing to determine the mechanisms. Finally, it is important to keep in mind that intermittent dosing is not a cure and drug cycling eventually will prove futile. Such strategy gives a patient more time, however, delaying the need to change inhibitors, initiate combination cocktails, or a move to traditional therapies like chemotherapy or radiation, all of which may be significantly more toxic. In the ever-expanding arsenal of weapons against cancer, strategies exploiting oncogene overdose appear to hold promise, and in the case of intermittent dosing don't even require development of new drugs.

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...

Due to its extensive length, DNA is packaged into a protective chromatin structure known as the nucleosome. In order to carry out various cellular functions, nucleosomes must be disassembled, allowing access to the underlying DNA, and subsequently reassembled on completion of these processes. The assembly and disassembly of nucleosomes is dependent on the function of histone modifiers, chromatin remodelers and histone chaperones. In this review, we discuss the roles of an evolutionarily conserved histone chaperone known as the HIR/HIRA complex. In S. cerevisiae, the HIR complex is made up of the proteins Hir1, Hir2, Hir3 and Hpc2, which collectively act in transcriptional regulation, elongation, gene silencing, cellular senescence and even aging. This review presents an overview of the role of the HIR complex, in yeast as well as other organisms, in each of these processes, in order to give a better understanding of how nucleosome assembly is imperative for cellular homeostasis and genomic integrity. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

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