As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non–BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.

Key Points• Low doses of the BRAF inhibitor vemurafenib are highly effective in refractory hairy cell leukemia.• Abrogation of BRAF V600E-induced signaling was consistently seen with 240 mg of vemurafenib twice daily.The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n 5 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care. (Blood. 2016;127(23):2847-2855

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

There is remarkable heterogeneity in the clinical course and biological characteristics of patient subgroups with chronic lymphocytic leukemia (CLL). Mutations of key tumor suppressors (ATM, miR-15a/16-1 and TP53) have been identified in CLL, and these aberrations are important "drivers" of the disease and some of its clinical characteristics. While some mutations are associated with poor outcome [particularly del(17p) and TP53 mutation], others are linked to a favorable clinical course [e.g. del(13q) as sole aberration]. In addition to genetic aberrations, antigen drive and microenvironmental interactions contribute to the pathogenesis of CLL. How the genetic aberrations impact on the process of antigen drive or microenvironmental interactions is currently unclear. Our improved understanding of the biology and clinical course of specific genetic subgroups is beginning to be translated into more specific and targeted treatment approaches. As a result, genetic subgroups are treated in distinct protocols. This review summarizes the contribution of the microenvironment and the most important genetic aberrations in CLL and how our improved knowledge of the biology of CLL may translate into improved treatment results.

SummaryRecurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16Á3%, 16Á9%, 10Á7%). We found evidence for subclonal mutations in 67Á5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CLL.

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Mutations in the N-terminus of MED12 protein occur at high frequency in uterine leiomyomas and breast fibroepithelial tumours, and are frequently found in chronic lymphocytic leukaemia (CLL). MED12 mutations have been previously linked to aberrant Cyclin C-CDK8 kinase activity, but the exact oncogenic function in CLL is unknown. Here, we characterized MED12 mutations in CLL and identified recurrent mutations in 13 out of 188 CLL patients (6·9%), which clustered in the N-terminus. MED12 mutations were associated with unmutated IGHV (P = 0·024). Protein analysis of NOTCH1 in primary CLL samples revealed increased levels of NOTCH1 intracellular domain (NICD), the active form of NOTCH1, in the context of MED12 mutations. We found evidence that NICD is the target of Cyclin C-CDK8 kinase using a specific CDK8 inhibitor. In line with these findings, MED12 mutations were mutually exclusive to mutations in NOTCH1 in CLL, based on a meta-analysis of 1429 CLL patients (P = 0·011). Our results suggest that MED12 mutations may contribute to CLL pathogenesis by activating NOTCH signalling.