New scaffolds for type II JAK2 inhibitors overcome the acquired G993A resistance mutation.

Recurrent JAK2 alterations are observed in myeloproliferative neoplasms, B-cell acute lymphoblastic leukemia, and other hematologic malignancies. Currently available type I JAK2 inhibitors have limited activity in these diseases. Preclinical data support the improved efficacy of type II JAK2 inhibitors, which lock the kinase in the inactive conformation. By screening small molecule libraries, we identified a lead compound with JAK2 selectivity. We highlight analogs with on-target biochemical and cellular activity and demonstrate in vivo activity using a mouse model of polycythemia vera. We present a co-crystal structure that confirms the type II binding mode of our compounds with the "DFG-out" conformation of the JAK2 activation loop. Finally, we identify a JAK2 G993A mutation that confers resistance to the type II JAK2 inhibitor CHZ868 but not to our analogs. These data provide a template for identifying novel type II kinase inhibitors and inform further development of agents targeting JAK2 that overcome resistance.

Germ line DDX41 mutations define a unique subtype of myeloid neoplasms.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes' pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia.

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome, whole-exome, and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains and amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains and amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL. SIGNIFICANCE: This study reveals the major role of gains, amplifications, and mutations of EPOR and JAK2 in the pathogenesis of pure erythroleukemia. Their frequent response to ruxolitinib in patient-derived xenograft and cell culture models highlights a possible therapeutic role of JAK2 inhibition for erythroleukemia with EPOR/JAK2-involving lesions. This article is highlighted in the In This Issue feature, p. 369.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Genomic landscape of young ATLL patients identifies frequent targetable CD28 fusions.

Adult T-cell leukemia/lymphoma (ATLL) in Japan presents at a median age of 70 years and only 5% of patients are <50 years of age. We conducted RNA and targeted DNA sequencing of 8 ATLLs from Japanese patients <50 years of age and identified 3 (37.5%) with both CTLA4-CD28 and inducible costimulator (ICOS)-CD28 fusions. Mutations of PLCG1, PRKCB, and STAT3, which were frequent in other ATLL-sequencing studies, were not identified. Differential expression analysis identified the negative checkpoint molecule LAG3 as the most downregulated gene among cases with the fusions. Immunohistochemistry demonstrated expression of CD80 and CD86, the ligands for CTLA4 and CD28, on ATLL cells and tumor-associated macrophages, respectively. Expression of CTLA4-CD28 in Ba/F3 cells conferred cytokine-independent growth when cocultured with Raji cells that express CD80 and CD86. Growth was associated with recruitment of the p85 subunit of phosphatidylinositol 3-kinase to CTLA4-CD28 and phosphorylation of AKT and extracellular signal-regulated kinase. A CTLA4-blocking antibody reduced cytokine-independent growth in a dose-dependent manner. Together, these results suggest that young Japanese ATLL cases have a unique biology dependent on cell-nonautonomous interactions that drive CD28 signaling. Assessment for CD28 fusions and treatment with CTLA4 blockade should be considered in younger patients with relapsed/refractory ATLL.

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

Molecular pathogenesis of disease progression in MLL-rearranged AML.

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P < 0.005). Among these, mutations in Ptpn11 (p.G60R) and Braf (p.V637E) corresponded to those identified in human MLL-AML, while recurrent mutations affecting Msn (p.R295C) were observed only in mouse but not in human MLL-AML. Another mutated gene of interest was Gnb2 which was reported to be recurrently mutated in various hematological neoplasms. Gnb2 mutations (p.G77R) were significantly increased in clone size (P = 0.007) and associated with earlier leukemia onset (P = 0.011). GNB2 transcripts were significantly upregulated in human MLL-AML compared to MLL-negative AML (P < 0.05), which was supported by significantly increased Gnb2 transcript induced by MLL/AF9 overexpression (P < 0.001). In in vivo model, both mutation and overexpression of GNB2 caused leukemogenesis, and downregulation of GNB2 expression reduced proliferative potential and survival benefit, suggesting a driver role of GNB2. In conclusion, alterations of driver genes over time may play an important role in the progression of MLL-AML.

Tumor-suppressor genes that escape from X-inactivation contribute to cancer sex bias.

There is a striking and unexplained male predominance across many cancer types. A subset of X-chromosome genes can escape X-inactivation, which would protect females from complete functional loss by a single mutation. To identify putative 'escape from X-inactivation tumor-suppressor' (EXITS) genes, we examined somatic alterations from >4,100 cancers across 21 tumor types for sex bias. Six of 783 non-pseudoautosomal region (PAR) X-chromosome genes (ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3) harbored loss-of-function mutations more frequently in males (based on a false discovery rate < 0.1), in comparison to zero of 18,055 autosomal and PAR genes (Fisher's exact P < 0.0001). Male-biased mutations in genes that escape X-inactivation were observed in combined analysis across many cancers and in several individual tumor types, suggesting a generalized phenomenon. We conclude that biallelic expression of EXITS genes in females explains a portion of the reduced cancer incidence in females as compared to males across a variety of tumor types.

Differential Regulation of the Melanoma Proteome by eIF4A1 and eIF4E.

Small molecules and antisense oligonucleotides that inhibit the translation initiation factors eIF4A1 and eIF4E have been explored as broad-based therapeutic agents for cancer treatment, based on the frequent upregulation of these two subunits of the eIF4F cap-binding complex in many cancer cells. Here, we provide support for these therapeutic approaches with mechanistic studies of eIF4F-driven tumor progression in a preclinical model of melanoma. Silencing eIF4A1 or eIF4E decreases melanoma proliferation and invasion. There were common effects on the level of cell-cycle proteins that could explain the antiproliferative effects in vitro Using clinical specimens, we correlate the common cell-cycle targets of eIF4A1 and eIF4E with patient survival. Finally, comparative proteomic and transcriptomic analyses reveal extensive mechanistic divergence in response to eIF4A1 or eIF4E silencing. Current models indicate that eIF4A1 and eIF4E function together through the 5'UTR to increase translation of oncogenes. In contrast, our data demonstrate that the common effects of eIF4A1 and eIF4E on translation are mediated by the coding region and 3'UTR. Moreover, their divergent effects occur through the 5'UTR. Overall, our work shows that it will be important to evaluate subunit-specific inhibitors of eIF4F in different disease contexts to fully understand their anticancer actions. Cancer Res; 77(3); 613-22. ©2016 AACR.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals.

Activity of the Type II JAK2 Inhibitor CHZ868 in B Cell Acute Lymphoblastic Leukemia.

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.

Targeting oncogenic interleukin-7 receptor signalling with N-acetylcysteine in T cell acute lymphoblastic leukaemia.

Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T-ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R-mutant DND-41 cells as assessed by non-reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND-41 cells, and Ba/F3 cells transformed by an IL7R-mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND-41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T-ALL.

Mutations in G protein ß subunits promote transformation and kinase inhibitor resistance.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gß subunits have not been defined. Here we demonstrate that recurrent mutations in the Gß proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gßγ dimer. Different mutations in Gß proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 Lys27 trimethylation.

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.

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.

Next-generation cDNA screening for oncogene and resistance phenotypes.

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

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.

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.

Molecular ontogeny of donor-derived follicular lymphomas occurring after hematopoietic cell transplantation.

UNLABELLED: The relative timing of genetic alterations that contribute to follicular lymphoma remains unknown. We analyzed a donor-recipient pair who both developed grade 2/3A follicular lymphoma 7 years after allogeneic transplantation and donor lymphocyte infusions. Both patients harbored identical BCL2/IGH rearrangements also present in 1 in 2,000 cells in the donor lymphocyte infusion, and the same V(D)J rearrangement, which underwent somatic hypermutation both before and after clonal divergence. Exome sequencing of both follicular lymphomas identified 15 shared mutations, of which 14 (including alterations in EP300 and KLHL6) were recovered from the donor lymphocyte infusion by ultra-deep sequencing (average read coverage, 361,723), indicating acquisition at least 7 years before clinical presentation. Six additional mutations were present in only one follicular lymphoma and not the donor lymphocyte infusion, including an ARID1A premature stop, indicating later acquisition during clonal divergence. Thus, ultrasensitive sequencing can map clonal evolution within rare subpopulations during human lymphomagenesis in vivo. SIGNIFICANCE: For the first time, we define the molecular ontogeny of follicular lymphoma during clonal evolution in vivo. By using ultrasensitive mutation detection, we mapped the time-course of somatic alterations after passage of a malignant ancestor by hematopoietic cell transplantation.