In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a ...particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.
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.
Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs ...targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.
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.
Recent evidence suggests that - in addition to 17p deletion - TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective ...clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4-9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.
The tumor microenvironment is the cellular and molecular environment in which the tumor exists and with which it continuously interacts. In B-cell lymphomas, this microenvironment is intriguing in ...that it plays critical roles in the regulation of tumor cell survival and proliferation, fostering immune escape as well as the development of treatment resistance. The purpose of this review is to summarize the proceedings of the Second Annual Summit on the Immune Microenvironment in Hematologic Malignancies that took place on September 11-12, 2014 in Dublin, Ireland. We provide a timely overview of the composition and biological relevance of the cellular and molecular microenvironment interface and discuss the role of interactions between the microenvironment and neoplastic cells in a variety of B-cell lymphomas. In addition, we focus on various novel therapeutic strategies that target the tumor microenvironment, including agents that modulate B-cell receptor pathways and immune-checkpoints, chimeric antigen receptor T cells and immunomodulatory agents.
T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. ...The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.
Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of ...disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.
Chronic lymphocytic leukemias (CLLs) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce ...interleukin 10 (IL-10). The two subsets differ in their levels of anergy, defined by low surface immunoglobulin M levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy. DNA methylation analysis of IL10 locus revealed two previously uncharacterized 'variably methylated regions' (CLL-VMRs1/2) in the gene body, but similarly low methylation in the promoter of both U-CLL and M-CLL. CLL-VMR1/2 methylation was lower in M-CLL than in U-CLL and inversely correlated with IL-10 induction. A functional signal transducer and activator of transcription 3 (STAT3) binding site in CLL-VMR2 was confirmed by proximity ligation and luciferase assays, whereas inhibition of SYK-mediated STAT3 activation resulted in suppression of IL10. The data suggest epigenetic control of IL-10 production. Higher tumor load may compensate the reduced IL-10 production in U-CLL, accounting for clinical immunosuppression in both subsets. The observation that SYK inhibition also suppresses IL-10 provides a potential new rationale for therapeutic targeting and immunological rescue by SYK inhibitors in CLL.
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