Conversion of the LIMA1 tumour suppressor into an oncogenic LMO-like protein by API2-MALT1 in MALT lymphoma.

MALT1 is the only known paracaspase and is a critical mediator of B- and T-cell receptor signalling. The function of the MALT1 gene is subverted by oncogenic chimeric fusions arising from the recurrent t(11;18)(q21;q21) aberration, which is the most frequent translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. API2-MALT1-positive MALT lymphomas manifest antibiotic resistance and aggressive clinical behaviour with poor clinical outcome. However, the mechanisms underlying API2-MALT1-induced MALT lymphomagenesis are not fully understood. Here we show that API2-MALT1 induces paracaspase-mediated cleavage of the tumour suppressor protein LIMA1. LIMA1 binding by API2-MALT1 is API2 dependent and proteolytic cleavage is dependent on MALT1 paracaspase activity. Intriguingly, API2-MALT1-mediated proteolysis generates a LIM domain-only (LMO)-containing fragment with oncogenic properties in vitro and in vivo. Importantly, primary MALT lymphomas harbouring the API2-MALT1 fusion uniquely demonstrate LIMA1 cleavage fragments. Our studies reveal a novel paracaspase-mediated oncogenic gain-of-function mechanism in the pathogenesis of MALT lymphoma.This work was supported in part by NIH grants R01 DE119249 and R01 CA136905 (K.S.J.E-J.), R01 CA140806 (M.S.L.) and the Department of Pathology at the University of Michigan.This is the accepted manuscript. The final version is available from Nature at http://www.nature.com/ncomms/2015/150108/ncomms6908/full/ncomms6908.html

A single center phase II study of ixazomib in patients with relapsed or refractory cutaneous or peripheral T‐cell lymphomas

The transcription factor GATA‐3, highly expressed in many cutaneous T‐cell lymphoma (CTCL) and peripheral T‐cell lymphomas (PTCL), confers resistance to chemotherapy in a cell‐autonomous manner. As GATA‐3 is transcriptionally regulated by NF‐κB, we sought to determine the extent to which proteasomal inhibition impairs NF‐κB activation and GATA‐3 expression and cell viability in malignant T cells. Proteasome inhibition, NF‐κB activity, GATA‐3 expression, and cell viability were examined in patient‐derived cell lines and primary T‐cell lymphoma specimens ex vivo treated with the oral proteasome inhibitor ixazomib. Significant reductions in cell viability, NF‐κB activation, and GATA‐3 expression were observed preclinically in ixazomib‐treated cells. Therefore, an investigator‐initiated, single‐center, phase II study with this agent in patients with relapsed/refractory CTCL/PTCL was conducted. Concordant with our preclinical observations, a significant reduction in NF‐κB activation and GATA‐3 expression was observed in an exceptional responder following one month of treatment with ixazomib. While ixazomib had limited activity in this small and heterogeneous cohort of patients, inhibition of the NF‐κB/GATA‐3 axis in a single exceptional responder suggests that ixazomib may have utility in appropriately selected patients or in combination with other agents.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139920/1/ajh24895.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139920/2/ajh24895_am.pd

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Survival following salvage therapy for primary refractory peripheral T‐cell lymphomas (PTCL)

Optimal salvage therapy for primary refractory peripheral T‐cell lymphomas (PTCL) and the role of hematopoietic stem cell transplant (SCT) remain poorly defined. We conducted a retrospective review of clinical outcomes and prognostic factors in a single‐center cohort of 93 patients with primary refractory PTCL, defined as progression during first‐line therapy or relapse within 6 months of its completion. Clinical outcomes were poor in this population, with median event‐free survival (EFS) of 3.5 months, median overall survival (OS) of 9.1 months, and 34% 3‐year survival. Outcomes were comparable in patients who progressed through first‐line therapy and patients who achieved CR/PR and subsequently relapsed within 6 months. A majority exhibited high‐risk features and had intermediate to high risk IPI, which correlated with inferior outcomes. There was no difference in outcomes between patients who received single‐agent salvage regimens and patients who underwent traditional, multi‐agent salvage regimens. Thus, participation in well‐designed clinical trials should be encouraged in this population. Additionally, there may be a trend toward improved EFS and OS in patients who underwent autologous or allogeneic SCT compared to patients who achieved CR or PR but were not transplanted.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142498/1/ajh24992.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142498/2/ajh24992_am.pd

Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare

T-Cell Receptor Signaling Activates an IKT/NF-κB/GATA-3 Axis in T-Cell Lymphomas Facilitating Resistance to Chemotherapy

Purpose: T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T cell-specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR\u27s role in mediating resistance to chemotherapy. Experimental Design: Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following TCR engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results: Here, we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance. Conclusions: These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented the activation of this signaling axis and overcame chemotherapy resistance