Diederik van Bodegom

A targeted mutational landscape of angioimmunoblastic T-cell lymphoma

The genetics of angioimmunoblastic T-cell lymphoma (AITL) are very poorly understood. We defined the mutational landscape of AITL across 219 genes in 85 cases from the United States and Europe. We identified ≥2 mutations in 34 genes, nearly all of which were not previously implicated in AITL. These included loss-of-function mutations in TP53 (n = 4), ETV6 (n = 3), CCND3 (n = 2), and EP300 (n = 5), as well as gain-of-function mutations in JAK2 (n = 2) and STAT3 (n = 4). TET2 was mutated in 65 (76%) AITLs, including 43 that harbored 2 or 3 TET2 mutations. DNMT3A mutations occurred in 28 (33%) AITLs; 100% of these also harbored TET2 mutations (P < .0001). Seventeen AITLs harbored IDH2 R172 substitutions, including 15 with TET2 mutations. In summary, AITL is characterized by high frequencies of overlapping mutations in epigenetic modifiers and targetable mutations in a subset of cases.

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.

TSLP signaling pathway map: a platform for analysis of TSLP-mediated signaling

Thymic stromal lymphopoietin (TSLP) is a four-helix bundle cytokine that plays a critical role in the regulation of immune responses and in the differentiation of hematopoietic cells. TSLP signals through a heterodimeric receptor complex consisting of an interleukin-7 receptor α chain and a unique TSLP receptor (TSLPR) [also known as cytokine receptor-like factor 2 (CRLF2)]. Cellular targets of TSLP include dendritic cells, B cells, mast cells, regulatory T (Treg) cells and CD4+ and CD8+ T cells. The TSLP/TSLPR axis can activate multiple signaling transduction pathways including the JAK/STAT pathway and the PI-3 kinase pathway. Aberrant TSLP/TSLPR signaling has been associated with a variety of human diseases including asthma, atopic dermatitis, nasal polyposis, inflammatory bowel disease, eosinophilic eosophagitis and, most recently, acute lymphoblastic leukemia. A centralized resource of the TSLP signaling pathway cataloging signaling events is not yet available. In this study, we present a literature-annotated resource of reactions in the TSLP signaling pathway. This pathway map is publicly available through NetPath (http://www.netpath.org/), an open access signal transduction pathway resource developed previously by our group. This map includes 236 molecules and 252 reactions that are involved in TSLP/TSLPR signaling pathway. We expect that the TSLP signaling pathway map will provide a rich resource to study the biology of this important cytokine as well as to identify novel therapeutic targets for diseases associated with dysregulated TSLP/TSLPR signaling. Database URL: http://www.netpath.org/pathways?path_id=NetPath_24

BCL2 Suppresses PARP1 Function and Nonapoptotic Cell Death

BCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment. Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1 activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On the basis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together, our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis.

Differences in signaling through the B-cell leukemia oncoprotein CRLF2 in response to TSLP and through mutant JAK2

Approximately 10% of B-cell acute lymphoblastic leukemias (B-ALLs) overexpress the cytokine receptor subunit CRLF2, which may confer a poor prognosis. CRLF2 binds its ligand thymic stromal lymphopoietin (TSLP) as a heterodimer with IL7R. Subsets of CRLF2-overexpressing B-ALLs also have a gain-of-function CRLF2 F232C mutation or activating mutations in JAK2. Whether these mutant alleles confer differences in signaling has not been addressed. Through a domain mutation analysis, we demonstrate a distinct dependence on the CRLF2 intracellular tyrosine Y368 in signaling by CRLF2 F232C, but not signaling induced by TSLP or through CRLF2/mutant JAK2. In contrast, CRLF2 signaling in each context is strictly dependent on both the CRLF2 box1 domain and the intracellular tryptophan W286. Using a global quantitative analysis of tyrosine phosphorylation induced by TSLP, we previously identified TSLP-induced phosphorylation of multiple kinases implicated in B-cell receptor signaling, including Lyn, Btk, Hck, Syk, MAPK8, MAPK9, and MAPK10. We now demonstrate that cells dependent on CRLF2/mutant JAK2 have reduced phosphorylation at these targets, suggesting that the kinases promote TSLP-mediated proliferation but serve as negative regulators of CRLF2/mutant JAK2 signaling. Thus, targetable nodes downstream of CRLF2 differ based on the presence or absence of additional mutations in CRLF2 signaling components.

Abstract 433: Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3

Our goal is to identify oncogenic loci in regions of recurrent DNA copy number alterations in cancer. Constitutional trisomy 21 (Down syndrome) carries a 20-fold increased risk of B-ALL, and chr.21 gains are the most common acquired aneuploidy in B-ALL. Interstitial amplification in the chr.21q22 region (iAMP21) is also a recurrent finding in B-ALL and carries a poor prognosis. However, the gene(s) on chr.21 responsible for this association remain unclear. We studied the Ts1Rhr mouse, which carries germline triplication of 31 genes homologous to human chr.21q22. Chr.21q22 triplication was sufficient to promote B cell autonomous self-renewal and maturation defects, and cooperated with BCR-ABL or CRLF2 with JAK2 R683G to accelerate leukemogenesis. Chr.21q22 triplication also resulted in histone H3K27 hypomethylation at gene promoters, and the expression signature of triplicated B cells was enriched for genes targeted by polycomb repressor complex 2 (PRC2), which trimethylates H3K27. Thus, chr.21q22 triplication may deregulate B cell development by causing H3K27 hypomethylation at genes critical for progenitor cell growth. In support of this hypothesis, pharmacologic inhibition of PRC2 function was sufficient to confer self-renewal in wild-type B cells, while inhibition of H3K27 demethylases blocked self-renewal induced by chr.21q22 triplication. In three independent B-ALL cohorts, PRC2/H3K27 gene signatures distinguished leukemias with +21 from those without, validating the same biology in human disease. One of the 31 triplicated genes, HMGN1, encodes a nucleosome binding protein known to modulate chromatin structure and facilitate transcriptional activation. When we overexpressed HMGN1 in BaF3 proB cells, H3K27me3 decreased proportionally to the level of overexpression. We next knocked down each of the 31 triplicated genes with lentivirally-expressed shRNAs (5 per gene) and assessed the effects on growth of Ts1Rhr and wild-type primary B cells. Strikingly, Hmgn1 was the top scoring gene and all 5 hairpins targeting Hmgn1 were depleted in the assay. Finally, we studied transgenic mice (HMGN1_OE) that overexpress human HMGN1 (∼2-fold total overexpression). HMGN1_OE mice had a defect in B cell maturation, increased proB colony forming capacity, and a transcriptional signature overlapping with that of triplication of all 31 Ts1Rhr genes. In a bone marrow transplant model driven by BCR-ABL, recipients of HMGN1_OE bone marrow developed B-ALL with decreased latency (median 33 days vs not reached) and increased penetrance (17/18 vs 4/17 mice died by 80 days; leukemia-free survival difference P Citation Format: Andrew A. Lane, Bjoern Chapuy, Charles Y. Lin, Trevor Tivey, Hubo Li, Elizabeth Townsend, Diederik van Bodegom, Tovah A. Day, Shuo-Chieh Wu, Huiyun Liu, Akinori Yoda, Gabriela Alexe, Anna Schinzel, Timothy J. Sullivan, Sebastien Malinge, Jordan Taylor, Kimberly Stegmaier, Jacob Jaffe, Michael Bustin, Geertruy te Kronnie, Shai Izraeli, Marian Harris, Kristen Stevenson, Donna Neuberg, Lewis B. Silverman, Steven E. Sallan, James E. Bradner, William C. Hahn, John D. Crispino, David Pellman, David M. Weinstock. Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2014-433