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Previous studies by us and others have linked germline genetic variants to the familial predisposition to childhood B-cell acute lymphoblastic leukemia (B-ALL), with pathogenic variants discovered in TP53, PAX5, ETV6, and IKZF1 (J. Clin. Oncol 2018, Nature Genet 2014, Lancet Oncol 2015, Cancer Cell 2018). However, genetic predisposition to T-ALL is much less understood. Rare care reports of T-ALL pedigrees with germline RUNX1 point to its potential role in ALL susceptibility. RUNX1 plays significant roles in definitive hematopoiesis and primarily functions as a transcription factor. RUNX1 germline variants are associated with familial platelet disorder, with a significant proportion of patients also developing myeloid malignancies. To comprehensively examine the pattern and prevalence of RUNX1 germline variation in T-ALL, we performed targeted germline sequencing of 1,231 cases enrolled on the Children's Oncology Group AALL0434 trial. In this largely unbiased T-ALL cohort, we identified 13 germline RUNX1 variants in 16 cases (Figure 1), including six missense (46.2%), two nonsense (15.4%), three frameshift (23.1%), and two indel variants (15.4%). These variants are divided into three groups: Group I, truncating both the DNA-binding RHD domain and the transcriptional activation AD domain (p.K117* and p.S141fs); Group II, truncating the AD domain only (p.Q213fs, p.R232fs, and p.Y287*); and Group III, missense and indel variants. To comprehensively characterize the function of these T-ALL-related RUNX1 variants, we performed a variety of biochemical and cellular assays in different model systems. Using reporter gene assays, we first directly evaluated the transcriptional activity of RUNX1 variants in Hela cells and identified both loss-of-function (e.g., Group I variants) and dominant-negative effects (e.g., p.G365R in Group III variants). Group I variants also showed dramatic subcellular mislocalization in the cytoplasm, with concomitant loss of CBFβ binding, both of which were significantly subtler for Groups II and III variants. Focusing on representative variants in these three groups (p.S141fs, p.R232fs, Y287*, and p.G365R), we next examined their effects on hematopoietic phenotypes in vitro. Ectopic expression of Group II and III variants in human CD34+ cells significantly increased CFU-M/GM colony formation and long-term proliferation, while repressing BFU-E colonies. Variant RUNX1 cells also showed defects in megakaryocyte and pre-T cell differentiation, with decreased apoptosis compared to cells expressing wild-type RUNX1. Expression of Group I variant led to phenotypes similar to that of empty vector, suggesting a complete loss of RUNX1 function. In parallel, we engineered isogenic T-ALL single clones with epitope-tagged RUNX1 variant introduced at the endogenous locus via CRISPR-Cas9 mediated homology recombination. Chromatin immunoprecipitation (ChIP)-seq profiling of these cells suggested a varying degree of changes in RUNX1 binding sites across the genome as a result of the RUNX1 genetic variation. On the other hand, RNA-seq profiling identified down-regulation of genes that were activated by wild-type RUNX1, again confirming the loss-of-function effects of these variants. Finally, we performed whole-genome seq of matched leukemia and germline samples and RNA-seq of leukemia cells in 7 T-ALL cases with RUNX1 predisposition variants. In this analysis, we observed a significant enrichment of JAK3 mutations (5 of 7 cases, 71.4%) compared to a cohort of 264 T-ALL with wild-type RUNX1 in the germline (P=3.39×10-7). By comparison, only 27.3% (3 of 7) of T-ALL with a somatic mutation in RUNX1 had concurrent JAK3 mutations in this cohort. Unsupervised clustering based on RNA-seq derived gene expression profile showed that RUNX1-mutated cases, either germline or somatic, clustered tightly with early T precursor (ETP) and near-ETP immunophenotypes. In conclusion, we comprehensively characterized 13 RUNX1 germline variants in T-ALL, ~40% of which are frameshift or nonsense. These variants result in a loss of function, by disrupting DNA binding or deleting the transcriptional activation domain, and in some cases in a dominant-negative fashion. RUNX1 genetic variation also results in significant defects in hematopoietic cell differentiation and functions in vitro, but additional somatic lesions are most likely required for overt leukemogenesis. Disclosures Gastier Foster: Incyte Corporation: Other: Commercial Research; Bristol Myers Squibb (BMS): Other: Commercial Research. Raetz:Pfizer: Research Funding. Zweidler-McKay:ImmunoGen: Employment. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Jazz: Honoraria. Relling:Servier Pharmaceuticals: Research Funding. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8+ T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+CD8+ cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P5 is the predominant S1P receptor in leukemic LGLs, whereas S1P1 is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and the etiology of this aggressive cancer is not fully understood. Common germline polymorphisms in lymphoid development genes and tumor suppressor genes have been associated with ALL susceptibility, although most have modest effects. Only a small fraction of ALL cases are thought to be related to congenital genetic disorders and consequently hereditary predisposition is rarely considered in clinical practice. However, a growing number of rare germline genetic mutations have been discovered in familial ALL (e.g., PAX5 , TP53 ), raising the possibility that the proportion of ALL attributable to inherited predisposition may be higher than currently proposed. In particular, germline ETV6 variations were recently reported in families with hereditary thrombocytopenia and dramatically increased susceptibility to hematologic malignancies ( Nat Genet 2015 47: 180 and 535). ETV6 is a transcriptional repressor essential for hematopoiesis and is frequently targeted by somatic genomic aberrations in childhood ALL (e.g., the ETV6 - RUNX1 fusion). Therefore, we sought to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which these variants contribute to childhood ALL risk in general. We first identified a family with three cases of childhood ALL at St. Jude Children9s Research Hospital. Whole exome sequencing of this family (mother and 2 daughters with ALL, the unaffected father and 1 unaffected daughter) identified a single variant in ETV6 (p.R359X) in the 3 cases with ALL and also in the healthy daughter. This nonsense variant is predicted to create a stop codon within the ETS domain of ETV6 , resulting in a truncated protein without DNA-binding function. This highly damaging variant is likely to be responsible for the ALL predisposition in this family with a high albeit incomplete penetrance. To comprehensively determine the prevalence of ALL-predisposing alleles in ETV6 , we performed targeted sequencing of this gene in 4,405 children with newly-diagnosed ALL enrolled on the Children9s Oncology Group (COG) AALL0232, P9904, P9905 and P9906 protocols and St. Jude Total Therapy XIIIA, XIIIB and XV studies. We identified a total of 43 germline variants in the exonic regions of ETV6 . Thirty-one of the 43 ETV6 variants were defined as "ALL-related" because they were not found or extremely rare in non-ALL populations (N=60,706). These ALL risk variants included 4 nonsense, 21 missense, 1 splice site, and 5 frameshift variants occurring in 35 children (0.79% of ALL cases studied). Fifteen of the 31 ALL-relatedvariants (48.4%) were clustered in the ETS DNA-binding domain of ETV6 . We used the combined annotation dependent depletion algorithm (CADD) to predict deleterious effects of each variant. ALL-related ETV6 variants were significantly more likely to be damaging compared to germline variants observed in the non-ALL population (mean CADD phred-like score of 25.6 vs 15.2, respectively, p ETV6 variants, 10 (55.6%) resided in the ETS domain although none were located within the helix directly interacting with target DNA. Instead, 7 of the 10 variants in ETS domain were between the first and second helices. We next analyzed the relationship between germline risk variants in ETV6 and clinical features of ALL in a subset of 2,021 cases enrolled on St. Jude and COG frontline ALL trials. These cases were comprehensively evaluted for ALL charateristics and representative of the US childhood ALL population. Children with ALL-related ETV6 variants were significantly older at the time of diagnosis than those without these variants (9.5 years vs 6.4 years; P=0.009). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (64.3% vs 26.8%; P=0.0045). In contrast, the frequency of somatic ETV6 - RUNX1 fusion was much lower in cases with ETV6 germline risk variants, compared to cases with wildtype ETV6 (7.1% vs 22.7%), even though this difference did not reach statistical significance. Of note, there was also a trend towards overrepresentation of females in carriers of ALL-related ETV6 variants (71.4% vs 45.7%; P=0.063). In conclusion, our findings indicate that germline ETV6 variations are important determinants for genetic predisposition to childhood ALL. Disclosures Martin: Jazz Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Gentium SpA/Jazz Pharmaceuticals: Research Funding. Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger: Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Merck: Equity Ownership; Sigma Tau: Consultancy.

While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Acute myeloid leukemia (AML) represents a genetically heterogeneous hematological malignancy and is among top 10 common cancers in China. Though most cases achieve complete remission with current therapy, relapses eventually occur and subsequent therapies fail to eliminate the leukemic cells again and sustain long-term remission. Acquired resistance might be the real instigator of treatment failure. Nuclear factor kappa B (NF-κB) signaling pathway activation, a hallmark of primary AML cells, especially of leukemic stem cells (LCSs) and in vitro cell lines, and associated with multi-layered roles in AML pathogenesis, i.e., pre-leukemia myelodysplastic syndrome (MDS), LSCs, drug response/toxicity, relapse, and leukemic maintenance. Thus, NF-κB might be an attractive strategy for better treatment response and survival but less toxicity in AML therapy. METHODS: Luciferase reporter assays were performed to define the effects of fenretinide on transcriptional signaling pathways. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the NF-κB down-stream genes expression, and immunoblotting was performed to confirm the role of fenretinide on NF-κB inihition and apoptosis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assay were performed to test the drug response and chemosensitizing effect of fenretinide on AML cell lines and primary AML samples. RESULTS: Initially, using series of specific reporter assay kits we experimentally evaluated the effects of fenretinide on cell signaling in HEK293T cells and ten leukemic cell lines. Among all tested signaling, we found that fenretinide-treated could significantly suppress the NF-κB activation induced by TNF-α in HEK293T cells and daunorubin (DNR)-induced NF-κB activationin AML cell lines. To molecularly confirm NF-κB suppression, we found that anti-apoptotic gene BCL2 was decreased and pro-apoptotic genes cIAP, XIAP, BID were increased. Also, immunoblotting showed the decreased protein level of p65 NF-κB in accompanied by increased level of cleaved-PARP, and BID, while no alteration of JNK, ERK proteins. Next, we went on testing the fenretinide-induced NF-κB inhibition in AML chemosensitizing using series of MTT and flow cytometric apoptotic assays. In this regard, we found that even low dose of fenretinide could chemosensitize AML cells to DNR treatment. Mechanistic studies showed that while ROS maintenance successfully rendered AML cells sensitive to DNR treatment, abolishing ROS production using N-Acetyl Cysteine (NAC) could not reversed the response of AML cells to DNR. In the meanwhile, NF-κB inhibition was the main cause of fenretinide-induced AML chemosensitizing. Finally, we made use of nine primary AML samples and then treated with fenretinide and/or DNR. In general, fenretinide could suppress NF-κB signaling but the inhibition effects varied between patients. Acutally, adding fenretinide indeed substantially potentiated the effect of DNR on AML cells. Also, the chemosensitizing effect was correlated with the level of fenretinide-induced NF-κB inhibition and the MDR1 gene down-regulation. CONCLUSIONS: The significance in our study is to identify the role of fenretinide-induced NF-κB inhibition in AML chemosensitizing through systemic in vitro experiments. Using in vitro AML cell lines and primary samples, we found that fenretinide could suppress NF-κB signaling pathway and sensitize AML cells to DNR treatment via reducing the pro-apoptotic/apoptotic genes expression, affecting proliferation associated genes expression and down regulating MDR1 expression. Low dose DNR in combination with low dose fenretinide produced similar effect comparable to that of high dose DNR treatment. Further, we identified that NF-κB inhibition but not ROS induction was the main reason for fenretinide-induced chemosensitizing on AML cells. These entire results highlight that fenretinide is a very promising chemosensitizing agent that would be of help in AML therapy. Disclosures No relevant conflicts of interest to declare.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. Proto-oncogene Platelet derived growth factor (PDGF) is a multifunctional molecule with roles in cell growth, proliferation, chemotaxis, actin reorganization and apoptosis resistance. PDGF exerts its cellular effects by inducing homo- or hetero-dimeric complex of α- or β-tyrosine kinase receptors, mediating its receptor tyrosine kinase (PDGF-β RTK) auto-phosphorylation, and consequently initiating downstream survival signaling pathway activation cascades. The importance of PDGF ligand and receptor activities in antigenactivated lymphoid malignancies is illustrated by their expression that has only been observed in HTLV-1/2 –immortalized human T lymphocytes, but not in normal T or NK cells. Large granular lymphocyte leukemia (LGL) is similarly defined as an antigen-activated malignancy. Same data suggest that LGL leukemia may be associated with an HTLV–like retrovirus infection. Since the LGL leukemia antigen is postulated to originate from a retroviral immunogen with HTLV-like properties, we investigated the role of PDGF in T- and NK-LGL leukemia. We report that LGL leukemia cells from both T and NK subtypes simultaneously express PDGF-BB and its receptor-β at both mRNA and protein levels. PDGF-BB mediates PDGF-β RTK auto-phosphrylation and consequently initiates downstream survival pathway activation cascade. We observed that PI3 kinase, Src family kinase and their downstream PKB/Akt pathways are constitutively activated in PBMC from LGL leukemia patients. Concurrent inhibition of both PI3 kinase and Src kinase pathways results in an additive effectiveness in induction of apoptosis via inhibition of PKB/Akt and MEK/ERK phosphorylation. In conclusion, PDGF-BB and PDGF receptor-β are highly expressed in LGL leukemia cells. Constitutive expression of PDGF-BB isoform from LGL leukemia cells may possibly be the result of chronic retroviral infection or retroviral antigenic stimulation. Co-expression of PDGF-BB and its receptor-β plays a central role in mediating PDGF/RTK auto-phosphorylation, and its downstream survival pathway activation cascades via an autocrine regulatory mechanism in LGL leukemia.