ALBERT

Beschreibung: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non Hodgkin lymphomas with poor prognosis. Molecular and cytogenetic studies have shown a prominent role for kinase fusion oncogenes, mostly NPM-ALK, in ALK+ anaplastic large cell lymphomas (ALCLs) and ITK-SYK kinase in unspecified PTCLs. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed an integrated mutation analysis using whole exome sequencing (n=12) and RNAseq (n=35) data. This analysis identified 288 candidate coding somatic mutations in 268 genes including known recurrent mutations in the TET2, DNMT3A and IDH2 epigenetic factor genes and pointed to the FYN kinase gene as a new recurrently mutated oncogene in T-cell transformation. The FYN tyrosine kinase is, with LCK, the predominant SRC family kinase found in T lymphocytes and plays an important role in T-cell activation upon T-cell receptor (TCR) stimulation. FYN mutations in PTCL included a FYN L174R mutation detected in one AITL patient sample, a FYN R176C allele recurrently found in two PTCL NOS cases and a FYN Y531H mutation present in a PTCL NOS sample. Notably, each of these alleles are predicted to specifically disrupt the inhibition of FYN kinase activity by the C terminal SRC kinase (CSK). Thus, structure analysis of FYN and FYN mutant proteins predicted that FYN L174R and, most prominently, FYN R176C and FYN Y531H can disrupt the inhibitory interaction of the FYN SH2 domain with the CSK-phosphorylated Y531. Consistently, pull down assays using GST-FYN-SH2 recombinant proteins and biotinylated C-terminal FYN peptides encompassing Y531 showed abrogation of the interaction between FYN-SH2 and P-Y531 in each of these mutants. In agreement with these results, expression of FYN L174R, FYN R176C and FYN Y531H resulted in increased levels of FYN activation. Moreover, CSK expression effectively inhibited wild type FYN, but failed to abrogate FYN L174R, FYN R176C or FYN Y531H activation. In contrast, pharmacologic kinase inhibition with dasatinib, a multikinase inhibitor which blocks ABL1 and SRC kinases, effectively abrogated the activity of FYN L174R, FYN R176C and FYN Y531H mutant proteins and suppressed the growth of cells transformed via expression of activated FYN mutant alleles. Overall these results support an oncogenic role for FYN activating mutations in the pathogenesis of PTCL and support a role for SRC kinase inhibitors for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of aggressive non Hodgkin lymphomas with poor prognosis. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed whole exome sequencing of matched tumor and normal DNA samples from 12 PTCL patients including 6 PTCL not otherwise specified (PTCL-NOS) tumors, 3 angioimunoablastic (AITL) T-cell lymphomas, 2 nasal type NK-/T-cell lymphomas and one enteropathy-associated T-cell lymphoma (EATL). This analysis identified 288 candidate coding somatic mutations in 268 genes and a mean mutation load of 24 non synonymous mutations per sample (range 4 – 57). Among these we noted the presence of a recurrent heterozygous mutation in the RHOA small GTPase gene (RHOA G17V) present in two independent AITL samples and one PTCL NOS biopsy. Analysis of a broad and diverse panel of 126 PTCL samples identified the presence of the RHOA G17V allele in 32 samples with a high prevalence in AITL (24/36, 67%, P 〈 0.001) and PTCL NOS cases (8/44, 18%, P 〈 0.002). The RHOA protein belongs to the Rho family of small GTPases, a group of Ras-like proteins responsible for linking a variety of cell-surface receptors to different intracellular signaling proteins. As is the case for RAS and most other small GTPases, RHOA activation is mediated by guanine exchange factors (GEFs), which catalyze the switch of RHOA from an inactive GDP-bound to an active GTP-bound state. Thus, and to test the functional significance of the RHOA G17V mutation we analyzed the capacity of this mutant to load GTP. This analysis revealed that RHOA G17V fails to incorporate GTP in response to an activated GEF in vitro. Moreover, and consistent with its inability to bind GTP, RHOA G17V failed to interact with rhotekin, a RHOA effector protein that selectively interacts with the GTP-bound active form of RHOA. However and most notably, the lack of RHOA G17V activation is not the result of a defect in RHOA-GEF interaction as RHOA G17V pull down assays demonstrated effective binding of this mutant protein to activated GEF proteins in T-cells. Based on these results we proposed an inhibitory role for RHOA G17V via sequestration of active GEF proteins. Consistently, while forced activation of RHOA signaling by GFP-RHOA overexpression induced loss of adhesion and round cell morphology in HEK293T cells, transfection of GFP-RHOA-G17V induced increased elongation and cellular protrusions as result of RHOA inactivation. In addition, immunoflourescence analysis of actin stress fiber formation by RHOA demonstrated effective abrogation of RHOA mediated cytoskeleton remodeling in cells expressing RHOA G17V. Overall these results show novel insight on the genetic basis of PTCLs and demonstrate a prominent role for RHOA G17V in the pathogenesis of AITL via disruption of RHOA signaling. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 406 T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease in which genetic lesions coordinately affect cell proliferation, differentiation and survival of thymocytes. Although pediatric T-ALL has been extensively studied on the genomic level during the last decade, large scale genomic studies in adult T-ALL are sparse. Importantly, significant differences in treatment outcome are present between pediatric and adult T-ALL. Here we performed integrative analysis of gene expression, copy number alterations and mutation analysis on 58 adult T-ALL samples treated in the Eastern Cooperative Oncology Group (ECOG) E2993 protocol. Unsupervised and consensus clustering of microarray gene expression data in this series revealed the presence of 2 stable gene expression clusters resembling immature/ETP (n=30) and typical (n=28) T-ALL gene signatures. Indeed, gene set enrichment analysis (GSEA) confirmed that our ETP/immature adult T-ALL signature closely resembled the previously published ETP profiles. In this adult patient cohort, the immature/ETP patient samples were associated with poor outcome (5 year OS 36% for ETP and 58% for non ETP TALL, P=0.088). Additional supervised analyses revealed differential expression of ERAP2, a gene involved in the processing of MHC peptides, between early relapse and long time survivor adult T-ALL patients. Consistently low ERAP2 expression was associated poor prognosis in adult T-ALL (OS 26%, 36% and 78% for 1st 2nd and 3rd terciles respectively, P=0.016). In addition, absence of TCR gamma deletion (p=0.013) as well as the absence of biallelic CDKN2A/CDKN2B deletion (p=0.009) were shown to be poor prognostic markers in adult T-ALL. To gain additional insight in the biology of adult ETP T-ALL, we performed GSEA analysis using the immature/ETP gene expression profiles. This analysis revealed that the adult ETP T-ALL signature is more closely related to acute myeloid leukemia (AML) profiles as compared to ALL signatures. Given this, we hypothesized that myeloid specific genetic alterations might be uniquely present in ETP adult T-ALL. Mutation analysis of AML oncogenes and tumor suppressor genes revealed the presence of myeloid mutations targeting IDH1, IDH2, DNMT3A, FLT3 and N-RAS in 13/30 (43%) of adult ETP T-ALL cases, whereas only 1/28 (3.5%) typical adult T-ALL showed a mutation in N-RAS. Although these ETP adult T-ALL samples seem to have myeloid properties, they still retain T-lymphoid characteristics including activational NOTCH1 mutations in 12/30 (40%) of ETP adult T-ALLs. Of note, the myeloid specific genetic alterations were preferentially present in NOTCH1 wild type ETP adult T-ALL samples. Next, we used a systems biology approach to identify master regulators that might be able to drive the ETP signature identified in adult T-ALL. For this, we interrogated a T-ALL interactome that was constructed from gene expression profiles of 228 human T-ALL samples. This master regulator analysis identified MN1 as the key transcription factor driving the ETP signature. The MN1 gene was initially identified as fusion partner of TEL (ETV6) in patients with AML. Notably, MN1-TEL expression in multipotent progenitors induces both AML and T-ALL and mutation analysis of ETV6 in adult T-ALL revealed the presence of 10 ETV6 mutants (9 frameshift and 1 splice site mutation) in adult T-ALL. Analysis of bone marrow remission genomic DNA confirmed the somatic origin of ETV6 mutations (3 frameshift and 1 splice site) in each of the 4 patient samples with available material. Most notably, ETV6 mutations were exclusively present in the ETP subtype of adult T-ALL. In addition, 8/10 (80%) of ETV6 mutant ETP T-ALL cases were characterized mutations in NOTCH1, suggesting a specific interaction between the oncogenic NOTCH1 programs and the mutational loss of ETV6 in early T-cell transformation. In conclusion, our integrative genomic analyses in adult T-ALL unraveled a high prevalence of immature/early T-cell progenitor (ETP) T-ALLs, associates the pathogenesis of ETP-TALL with myeloid mutations and identifies ETV6 truncating alleles as the first ETP specific gene mutation that strongly interact with NOTCH1 in T-cell transformation. Disclosures: No relevant conflicts of interest to declare.