ALL Subtypes Research Articles

Association of genetic variation in IKZF1, ARID5B, and CEBPE and surrogates for early-life infections with the risk of acute lymphoblastic leukemia in Hispanic children.

Genome-wide association studies focusing on European-ancestry populations have identified ALL risk loci on IKZF1, ARID5B, and CEBPE. To capture the impacts of these genes on ALL risk in the California Hispanic population, we comprehensively assessed the variation within the genes and further assessed the joint effects between the genetic variation and surrogates for early-life infections (the presence of older siblings, daycare attendance, and ear infections). Genotypic data for 323 Hispanic ALL cases and 454 controls from the California Childhood Leukemia Study were generated using Illumina OmniExpress v1 platform. Logistic regression assuming a log-additive model estimated odds ratios (OR) associated with each SNP, adjusted for age, sex, and the first five principal components. In addition, we examined potential interactions between six ALL risk alleles and surrogates for early-life infections using logistic regression models that included an interaction term. Significant associations between genotypes at IKZF1, ARID5B, and CEBPE and ALL risk were identified: rs7780012, OR 0.50, 95% confidence interval (CI) 0.35-0.71 (p = 0.004); rs7089424, OR 2.12, 95% CI 1.70-2.65 (p = 1.16 × 10(-9)); rs4982731, OR 1.69, 95% CI 1.37-2.08 (p = 2.35 × 10(-6)), respectively. Evidence for multiplicative interactions between genetic variants and surrogates for early-life infections with ALL risk was not observed. Consistent with findings in non-Hispanic White population, our study showed that variants within IKZF1, ARID5B, and CEBPE were associated with increased ALL risk, and the effects for ARID5B and CEBPE were most prominent in the high-hyperdiploid ALL subtype in the California Hispanic population. Results implicate the ARID5B, CEBPE, and IKZF1 genes in the pathogenesis of childhood ALL.

IL2RA (CD25) Recruits Inhibitory Phosphatases to the Cell Membrane and Mediates Negative Feedback Control of STAT5 Signaling in Acute Lymphoblastic Leukemia

▪Background and hypothesis: CD25 (IL2RA, interleukin 2 receptor α chain) is a transmembrane protein with a 13aa cytoplasmic tail. CD25 cooperates with β- and γ-chains in binding IL-2, but does not contribute to cytokine signaling. During normal B cell development, CD25 is specifically upregulated on the surface of IL7-dependent pre-B cells and is also expressed on the surface of a subset of human pre-B ALL cases. CD25-expressing ALL is typically associated with poor clinical outcome. For these reasons, we studied the functional significance of CD25 expression on human pre-B ALL cells.Results: Flow cytometry and immunohistochemistry staining on a large panel of patient samples (n=416; MDACC, ECOG) revealed specific cell surface expression of CD25 in Ph+ ALL and Ph-like ALL, which are both high-risk subtypes of ALL. In agreement with selective expression on high-risk subsets, high expression levels of CD25 at the time of diagnosis were predictive of poor overall clinical outcome in these studies (P=0.005). BCR-ABL1 in Ph+ ALL and related tyrosine kinases in Ph-like ALL strongly activate STAT5, which then induces transcriptional activation of the IL2RA locus. Since Stat5 is also active during normal pre-B cell differentiation, we first analyzed B cell development in Il2ra-/- mouse bone marrow. Il2ra-/- B cell development was blocked at the pre-B cell stage, consistent with specific upregulation of CD25 on pre-B cells. In human Ph+ ALL cells, we found IL2RB and IL2RG were not co-expressed with CD25, suggesting a function of CD25 in Ph+ALL that is distinct from IL2 signaling. To test the biological significance of tyrosine kinase/STAT5-induced activation of CD25, we developed an Il2ra-/- mouse model for BCR-ABL1 pre-B ALL. Interestingly, the cytoplasmic tail of CD25 includes phosphorylation sites (S268 and T271) that are known substrates for serine/threonine phosphorylation by PKCα, which was reported to regulate protein phosphatase 2A (PP2A). To investigate interacting proteins with the cytoplasmic tail of CD25, we performed immune precipitation (IP) against the flag-tagged CD25-tail in primary Ph+ ALL cells which were transduced with either a CD25-tail-flag or an EV-flag vector. 2D mass spectrometry and Western blot on the IP products confirmed strong interactions with PKCα and PP2A. Weestern blot analysis confirmed additional interactions with inhibitory phosphatases including PTEN, PTPN6 (SHP1) and Inpp5d (SHIP1) in human Ph+ALL cells. In addition, both 2D MS and Westernblot showed recruitment of the Stat5-feedback inhibitors CISH, SOCS2 and SOCS3 at the CD25 cytoplasmic tail.Studying functional parameters of Il2ra-/-BCR-ABL1 ALL cells, we found impaired proliferation and colony formation capacity and drastically increased increased phosphorylation levels of pABLY412, pSTAT5Y694, pERKT202/Y204, pAKTS473, pP38T180/Y182 and p53. Reconstitution of CD25 expression restored normal phosphorylation levels of these molecules, as well as proliferation and colony formation.In a serial transplant setting, we observed that leukemia initiation in transplant recipients from Il2ra-/-BCR-ABL1 ALL cells required 10- to 100-times higher cell numbers, suggesting that CD25 contributes to leukemia initiation. In addition, CD25 expression is associated with a higher level of drug-resistance: In patient-derived pre-B ALL cells with mixed CD25Low and CD25High populations, the standard chemotherapy agent vincristine selectively induced apoptosis of in CD25Low but not CD25High ALL cells. An anti-CD25 immunotoxin drugs efficiently eradiated CD25High leukemia cells and thereby overcame drug-resistance against vincristine.Conclusions: Our studies identified CD25 as a surface receptor that mediates membrane recruitment of PP2A and CISH, SOCS2, negative feedback regulators of STAT5. CD25 is transcriptionally activated by STAT5 and therefore specifically expressed on high-risk ALL subtypes with oncogenic activation of the Stat5 pathway (Ph+ ALL and Ph-like ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.

Clinical Outcome of De Novo Adult Acute Lymphoblastic Leukemia (ALL) with 11q23/Mixed Lineage Leukemia (MLL) Gene Rearrangements

Introduction: The MLL gene on located on 11q23 plays an essential role in positive regulation of gene expression in early embryonic development and hematopoiesis. Fusion genes, such as MLL-AF4 resulting from t(4:11)(q21;q23), alter normal cellular proliferation and differentiation, favoring leukemogenesis. The DOT1L histone methyltransferase associated with MLL appears to be required for maintenance of ALL. MLL-AF4 is detected in about 10% of de novo B-lymphoblastic leukemia or 30=40% of pro-B ALL subtypes. The 11q23/MLL subtype of ALL has been associated with extremely poor outcomes. We conducted a retrospective analysis of de novo adult ALL with 11q23/MLL gene rearrangements.Methods: Overall the database included 74 pts with 11q23/MLL gene rearrangements referred to our institution between 1980 and 2014. Of these, 20 (27%) pts were relapsed/refractory, 4 (5%) were minimally pretreated, 5 (7%) were inevaluable for response, 3 (4%) had concomitant Philadelphia chromosome, 2 (3%) were T-lineage, 2 (3%) were mature B-ALL. The remaining 38 cases comprised the study cohort evaluated with respect to pretreatment characteristics and outcome measures such as response to therapy, remission duration (CRD), and survival (OS).Results: Baseline characteristics of the cohort included median age 44 yrs (range, 20-75); 26% were older than 60 yrs. Ten (26%) pts had a prior malignancy and were designated secondary ALL. Five (13%) pts had CNS disease at presentation. Median WBC count was 69.8 K/uL (range, 0.5-612); 24 (63%) pts had WBC > 30 K/uL. Nineteen (50%) pts had serum LDH > 1400 U/L. Co-expression of myeloid markers (CD13, CD33 +/- CD117) was noted in 6 (16%) pts. Distribution of the 11q23/MLL gene aberrancies were: t(4;11) (q21; q23) in 25 (66%) pts, 11q23 without translocation partner in 10 (26%) pts, and t(11;19)(q23;p13.1) in 3 (8%) pts. Thirty two (84%) pts were treated per the hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with high dose methotrexate and cytarabine) regimen (3 [10%] received concurrent rituximab) and 6 (16%) pts were treated per the augmented BFM regimen. Overall response rate was 95%; complete remission (CR) in 92% and CR without platelet recovery (CRp) in 3%. Overall median CRD was 26.3 mos (range 0.2-93+ mos); median OS was 24.2 mos. Overall, the 5-yr CRD and OS rates were 19% and 26%, respectively, with a median follow-up of 60 mos (range, 4.8-173+ mos). Median OS in pts with 11q23 without chromosomal translocation was 48.5 mos, compared with 24 mos for t(11; 19) and 13.3 mos for t(4; 11) (p= 0.07) (Fig. 1). There were no differences in outcomes by older age or designation as secondary ALL.Conclusion: The 11q23/MLL positive B-lymphoblastic subsets are associated with differentially inferior outcomes, although MLL-AF4 remains the ALL subset associated with highest risk of disease recurrence particularly in the absence of allogeneic stem cell transplantation; the relatively rarity of these subsets poses challenges in establishing the optimal treatment strategies. Therapeutic approaches incorporating hypomethylating agents, histone deacetylase inhibitors, and CAR-T cells could improve these dismal outcomes; targeted agents such as DOT1L inhibitors are under investigation in clinical trials. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL.Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling.Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation.Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells.Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. DisclosuresNo relevant conflicts of interest to declare.

BCR-ABL+ Bp-ALL Exhibits Heightened Sensitivity to Simultaneous Induction of Energy and ER Stress Via Downregulation of Mcl-1 Expression

Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients. DisclosuresNo relevant conflicts of interest to declare.

LNK (SH2B3) Synergizes with TP53 in Suppressing B-Precursor Acute Lymphoblastic Leukemia through IL-7 Receptor Signaling

B-precursor acute lymphoblastic leukemia (B-ALL) is the leading cause of cancer-related deaths in children and commonly has a poor outcome in adults. Gene profiling and exome sequencing of high-risk ALLs led to the recent identification of the Philadelphia chromosome (Ph)-like ALL subtype. These leukemias have gene expression profiles similar to BCR-ABL1-positive (Ph+) ALL, but lack a BCR-ABL1 rearrangement, and often result in poor outcomes. Many Ph-like ALL-associated mutations identified to date are known or predicted to activate oncogenic cytokine receptor signaling pathways, particularly those associated with JAK. The lymphocyte adaptor protein LNK (also called SH2B3) has emerged as a powerful negative regulator of cytokine-mediated JAK2 signaling in hematopoietic stem cells (HSCs). Loss-of-function LNK mutations and deletions have recently been described in B-ALL, and germline loss of LNK contributes to pediatric ALL development. However, the mechanisms by which LNK alterations impact leukemogenesis remain poorly understood. Here we show that LNK synergizes with TP53 and INK4a in suppressing B-ALL development in mice. Tp53-/-Lnk-/- mice developed highly aggressive and transplantable B-ALL with 100% penetrance in contrast to T-lymphoma or sarcoma development observed in Tp53-/- mice. Importantly, gene expression profiles of Tp53-/-Lnk-/- B-ALL blasts were similar to those of human Ph-like B-ALLs, validating the relevance of this model for preclinical studies. Tp53-/-Lnk-/- pro-B progenitors initiated B-ALL in the transplanted recipients, and Lnk loss-of-heterozygosity (LOH) was found exclusively in B-ALL cells from Tp53-/-Lnk+/- mice, but not in HSCs, common lymphoid progenitors, or cells of myeloid/T cell lineages, attesting that LNK is a bona fide tumor suppressor in the committed B-cell precursors. Mechanistically, we found that pre-leukemic Lnk-/-Tp53-/- pro-B progenitors were hypersensitive to IL-7 and showed markedly enhanced self-renewal ability in vitro and in vivo. Our genetic studies revealed that LNK controls normal B cell and B-ALL development independently of its effects on HSCs and specifically regulates pro-B cell homeostasis. A novel phosphoflow cytometry approach in freshly isolated BM cells that combines the surface marker B220, intracellular mu-heavy chain detection, and phospho-specific antibodies enabled separation of subpopulations of B progenitors with distinct IL-7 responsiveness. Using this approach, we demonstrated that LNK deficiency potentiated STAT5 activation in response to IL-7 in pre-leukemic pro-B cells. Of note, Lnk-/-p53-/- leukemic blasts with elevated pSTAT5, were less sensitive to JAK inhibitors than pre-leukemic B progenitors, in part due to constitutive activation of MAPK and AKT/mTOR pathways. Our results invoke the targeting of these pathways as novel therapeutic approaches in B-ALL. In sum, we have developed a novel B-ALL mouse model suitable for preclinical studies aimed at further deciphering the pathogenic mechanisms underlying this disease and exploring new therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Dual Targeting of JAK2 Signaling with a Type II JAK2 Inhibitor and of mTOR with a TOR Kinase Inhibitor Induces Apoptosis in CRLF2-Rearranged Ph-like Acute Lymphoblastic Leukemia

Philadelphia chromosome-like acute lymphoblastic leukemia (“Ph-like ALL”) is a subtype of high-risk B-precursor ALL (B-ALL) that carries a high risk of relapse after conventional chemotherapy (Mullighan et al, N Engl J Med. 2009). Rearrangements in CRLF2, leading to overexpression of the receptor for the cytokine thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALLs and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010; Tasian et al, Blood 2014). Previous studies established that combining a tyrosine kinase inhibitor (TKI) with an mTOR inhibitor provides greater anti-leukemia efficacy than a TKI alone in Ph+ B-ALL (Janes et al, Nat. Med. 2013). While allosteric mTOR inhibitors such as rapamycin only partially block mTORC1 and do not directly inhibit mTORC2, second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) efficiently block both mTOR outputs and show greater efficacy when combined with TKIs. In this study, we investigated anti-leukemia efficacy and intracellular signaling networks in Ph-like CRLF2+ ALL models treated with combinations of a type I or type II JAK-2 inhibitor and a TOR-KI.The inhibitors were tested in human B-precursor Ph-like ALL cell lines MUTZ5 (IGH@-CRLF2 translocation, JAK2 R683G mutation) and MHH-CALL-4 (IGH@-CRLF2 translocation, JAK2 I682F mutation), B-ALL cell line REH (CRLF2wt), and primary CRLF2+ xenograft cells in vitro. For signaling and growth inhibition studies, cells were stimulated with 25 ng IL-7 or TSLP for 30 min, then with JAK2 type I inhibitor ruxolitinib (500nM) or type II inhibitor NVP-BBT594 (500nM) (Andraos et al., Cancer Discov. 2012) and allosteric mTOR inhibitor rapamycin or TOR-KI AZD2014. Effects on intracellular signaling were determined by phospho-flow cytometry. Anti-leukemia effects were characterized by viable cell counts and annexin V flow cytometry.In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (p-JAK2(Tyr1008), p-STAT5(Ty694)) and AKT/pS6 signaling (p-AKT(Ser473), p-rS6(S235/236) (Fig. 1A). Stimulation with IL-7, mimicking support by the normal bone marrow environment, induced a lesser degree of activation of these phospho-proteins, except for p-4EBP1(T37/46), which was constitutively highly expressed in these cells and further induced by IL-7. These findings warranted combination studies of JAK2 and mTOR inhibitors. JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, and S6 activation, yet failed to decrease p-4EBP1 (Fig. 1A). AZD2014 but not rapamycin fully inhibited p-4EBP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth arrest in CRLF2+ cells (Fig. 1A, C). In turn, combination of ruxolitinib and AZD2014 further reduced cell proliferation but did not induce apoptotic cell death (Fig. 1B, D).Recent studies indicate persistence of JAK2-mutated cells in myeloproliferative neoplasms upon long-term exposure to a type I JAK2 inhibitor, mediated by JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2, in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of STAT5 phosphorylation; in contrast, BBT594 diminished both p-JAK2 and p-STAT5. Unexpectedly, BBT594 induced apoptotic cell death in both MUTZ5, MHH-CALL-4 (Fig 1B) and in ALL blasts recovered from primary CRLF2+ xenograft and grown in OP9 in vitro co-culture; the combination of BBT594 with AZD2014 increased apoptosis and reduced cell viability even further, in both cell lines and in stroma-attached primary ALL cells.In summary, these results suggest that efficient blockade of JAK2/STAT5 with a type II JAK2 inhibitor translates into cell death of JAK2-addicted CRLF2-rearranged cells and may have the capacity to eliminate JAK2-mutated clones. Concomitant blockade of TORC1 signaling with a TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4EBP1 and causes synthetic lethality, providing avenues for novel, rationally designed combinatorial regimens in this subset of Ph-like B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Clinical Features and Prognosis of Early T-Cell Precursor Subtype of Lymphoblastic Lymphoma in the Japanese Pediatric Leukemia/Lymphoma Study Group ALB-NHL03 Study

Abstract Introduction: Recently, early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) was identified as a subtype of T-cell ALL (T-ALL), with distinctive gene expression and cell marker profiles. Some reports revealed that ETP-ALL was associated with a high risk of remission induction failure and relapse. In precursor T-cell lymphoblastic lymphoma (T-LBL), the clinical features and prognosis of the ETP subtype are not clear yet. In this study, we analyzed the data obtained from patients of advanced stage T-LBL to clarify the prognosis of pediatric T-LBL according to the immunophenotypes, including the ETP subtype of LBL. Patients and methods: From November 2004 to October 2010, 136 children (aged 1–18 years) with newly diagnosed advanced stage LBL (stages III and IV) were eligible for the Japanese Pediatric Leukemia/Lymphoma Study Group ALB-NHL03 study. We analyzed their immunophenotyping data as well as the ETP subtype. The immunophenotype of T-LBL was classified into pro-T, pre-T, intermediate T, and mature T based on the European Group for the Immunological Characterization of Leukemias (EGIL) classification. The definition of ETP subtype LBL was based on a previous report from the Tokyo Children’s Cancer Study Group (Inukai et al, 2011) using a scoring system consisting of the following 11 markers: CD4, CD8, CD13, CD33, CD34, HLA-DR, CD2, CD3, CD4, CD10, and CD56. Both definitions were based on flow cytometric analysis. Results: In this analysis, 104 (76%) patients were diagnosed with T-LBL. Sufficient data to evaluate the EGIL classification was available for 40 out of 104 patients. The remaining patients could not be classified due to incomplete immunophenotypic data. There were 1, 9, 21, and 9 cases of Pro-T, pre-T, intermediate T, and mature T, respectively. The 3-year event-free survival (EFS) of pro-T/pre-T and intermediate T/mature T was 80.0 ± 12.7% and 76.7 ± 7.7%, respectively (P = 0.7586). For evaluating the ETP subtype of LBL, sufficient data, obtained by using the scoring system with 11 markers, was available for 40 patients. Eight (20%) and 32 (80%) patients were classified as having ETP and non-ETP subtype, respectively. Bone marrow involvement for patients with ETP and non-ETP subtype was observed in 7 (88%) and 11 (34%) cases, respectively (P = 0.014). Central nervous system involvement in patients with ETP and non-ETP subtype was observed in 2 (25%) and 0 cases, respectively (P = 0.036). Thus, stage IV classification was more frequently observed in patients with ETP subtype than in patients with non-ETP subtype (P = 0.014). The 3-year EFS of patients with ETP and non-ETP subtype were 75.0 ± 15.3% and 71.9 ± 8.0%, respectively. There was no significant difference in EFS between patients with ETP and non-ETP subtype (P = 0.8281). Conclusion: For 40 out of 104 T-LBL patients, sufficient data was available to evaluate the immunophenotype for EGIL classification and ETP subtype. There was no significant difference in EFS according to the immunophenotypic subtype of T-LBL. In contrast to T-ALL, ETP subtype in LBL was not statistically related to EFS in this analysis. Disclosures No relevant conflicts of interest to declare.

Detection of microvesicle miRNA expression in ALL subtypes and analysis of their functional roles.

Microvesicles (MVs) are the heterogeneous mixtures of vesicles. MVs released by leukemia cells constitute an important part of the leukemia microenvironment. MVs might act as important reservoirs of microRNAs (miRNAs). It is worth evaluating whether MVs possess some unique miRNA contents that are valuable in understanding the pathogenesis. In this study, we investigated the miRNA expression patterns of Nalm-6-derived MVs, Jurkat-derived MVs and normal cell-derived MVs using miRNA microarrays. The potential target genes regulated by differentially expressed miRNAs were also predicted and analyzed. Results demonstrated that 182 miRNAs and 166 miRNAs were differentially expressed in Nalm-6-MVs and Jurkat-MVs, respectively. Many oncogenes, tumor suppressors and signal pathway genes were targeted by these aberrantly expressed miRNAs, which might contribute to the development of B-ALL or T-ALL. Our findings expanded the potential diagnostic markers of ALL and provided useful information for ALL pathogenesis.

A Novel Subtype-Classifier Of Pediatric Acute Lymphoblastic Leukemia Using Gexp Multiplexed System

▪ ObjectiveAcute lymphoblastic leukemia (ALL) is the most frequent malignant neoplasm in children. The lack of reasonable and accurate classification has becomes the main obstructive factor, which makes normative treatment more difficult to carry out. Morphology, immunology, cytogenetics and molecular biology (MICM) classification is widely used clinically for pediatric leukemia. However, it is a time-consuming and expensive process and only available in a few major medical centers in some developing countries, which heavily impact the accurate classification. Our previous microarray analysis of gene expression profiles in 100 pediatric ALL bone marrow samples screened out 62 classification markers (mapped to 61 ENTREZ genes), which could classify pediatric ALL into 6 major ALL subtypes. In this study, the GenomeLab Gene Expression Profiler Genetic Analysis System (GeXP) was applied here to custom design a multiplex of 57-gene classifier to validate the feasibility of these genes as classification markers, and then establish a new and practical method for ALL classification, which will guide the risk classification and stratified treatment of leukemia. MethodsSixty-two classification markers were divided into 3 panels with each panel containing 20∼21 genes. Each primer is a chimeric primer consists of a gene-specific sequence fused to a universal tag sequence at the 5' terminal. The GeXP technology was used to measure the expression levels of these genes. The peak areas of each target gene and endogenous reference controls were normalized, from which, the relative quantification of each gene in a sample was determined. For each sample, we ranked the gene expression values from low to high and normalized these rank values by Z-score. Super k-means method was used to do the cluster by genes and samples. ResultsA novel gene expression-based ALL subtype classification using GeXP multiplexed assay was optimized and developed, which led to a rapid, reliable, and cost-effective classifier. Using a subset of 61 genes, we totally got 57 marker genes' expression data in 85 pediatric ALL samples. From the cluster results, 1) we got 4 clusters that mainly represented by 4 subtypes of ALL, including TEL-AML1+ ALL, BCR-ABL+ ALL, E2A-PBX1+ ALL and T-cell ALL, which obtained a high accuracy (98.4%, 60/61) with MICM classification (Fig 1); 2) we found the marker genes for each subtype and especially for those samples without any subtype, which provided the prediction according to which cluster it located in; 3) this classifier could take a single sample and made a prediction based solely on the relative expression ranks among the marker genes. ConclusionsWe develop a novel multiplexed 57-gene classifier to identify the major subtypes of pediatric ALL. This promising molecular test allows for a high-throughput, robust and reproducible assessment of multiplexed gene expression analysis. It offers a powerful diagnostic tool for the rapid classification using a minimal amount of samples. It may be usefully applied in the future clinical work and assist the physician in a more rapid and accurate classification of the subtypes in pediatric ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Targeting Pre-B Cell Receptor and BCL6 In TCF3-PBX1 B-Lineage Acute Lymphoblastic Leukemia

▪ BackgroundTCF3-PBX1 is one of the most common recurrent translocations which define distinct subtypes of B-lineage Acute Lymphoblastic Leukemia (B-ALL). Patients with TCF3-PBX1 B-ALL have poor clinical outcome, however, the molecular mechanisms underlying poor outcome are poorly understood. It is critical to identify the cellular processes that contribute to the biological and clinical features of this form of ALL and to develop new targeted therapeutic strategies to improve the outcome. ResultsWe first performed ChIP-seq with antibodies against the fusion oncoprotein TCF3-PBX1 in the human B-ALL cell line 697 and found that the pre–B cell receptor (pre-BCR) genes (IGLL1 or λ5, VpreB, CD79A, CD79B) and µ-chain enhancer regions were directly bound by the fusion protein. Gene expression microarray data showed that the pre-BCR signaling genes (IGLL1, VpreB, IGHM, BLK, LCK, SYK, LYN, SRC and BLNK) were overexpressed in TCF3-PBX1, but not other cytogenetic subtypes of B-ALL (n=132 B-ALL patient samples, St. Jude), suggesting a unique high pre-BCR activity in TCF3-PBX1 ALL. Further flow cytometry analysis using µ-chain specific antibodies showed a strong Ca2+ signal in TCF3-PBX1, but not other subtypes of ALL (n=27). The sequencing analysis on IGHM locus in 148 primary B-ALL samples showed that 100% cases of TCF3-PBX1 (n=8) carried functional IGHM VHDJH gene rearrangements, however only 17% for BCR-ABL1 (n=57), 0% for MLL-AF4 (n=7), 31% for ETV6-RUNX1 (n=13), 3.3% for hyperdiploid (n=30), 20% for sporadic (n=20) and 31% for normal karyotype (n=13) ALL. These data demonstrates that TCF3-PBX1 ALL has an unusual spectrum of high activity of pre-BCR signaling.The transcriptional repressor BCL6 has been identified as a critical survival factor in diffuse large B-cell lymphoma. We found that BCL6 was highly expressed in TCF3-PBX1 as compared to other subtypes of ALL by our Western blot (n=15) and also by gene expression microarray data from three B-ALL clinical trials: COG P9906 (n=207), ECOG E2993 (n=191) and St. Jude ALL (n=132). The clinical data showed that high expression of BCL6 correlates with poor clinical outcome. Those data suggested BCL6 might play a critical oncogene role in TCF3-PBX1 ALL. The TCF3-PBX1 ChIPseq data showed no binding enrichment on BCL6 locus. However, forced expression of pre-BCR components (µ-chain and BLNK) resulted in up-regulation of BCL6 (Western blot), suggesting BCL6 upregulation in TCF3-PBX1 ALL was induced by pre-BCR but not the fusion protein binding. To further test this, we applied SYK and BTK inhibitors to treat TCF-PBX1 ALL cells and found that they dramatically decreased BCL6 mRNA and protein levels and also induced cell apoptosis, suggesting BCL6 might be a therapeutic target for TCF3-PBX1 ALL. To test this, we transduced a primary TCF3-PBX1 B-ALL xenograft sample with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell death. When treating primary TCF3-PBX1 ALL cells with the specific BCL6 peptide inhibitor RI-BPI and the small molecule inhibitor PU-H71, they significantly induced cell cycle arrest, compromised colony formation, and prevented leukemia-initiation in transplant recipient mice. Collectively, those data indicates BCL6 is a new therapeutic target for TCF-PBX1 ALL.Dasatinib is a second generation tyrosine kinase inhibitor (TKI) targeting both BCR-ABL1 and Src kinase. We hypothesized that TCF3-PBX1 ALL cells would be more sensitive to Dasatinib than other TKIs because Dasatinib inhibits pre-BCR signaling and hence BCL6. We treated primary TCF3-PBX1 B-ALL cells with Dasatinib and Nilotinib, and found that BCL6 protein expression was abolished with Dasatinib, but no changes with Nilotinib. We also observed a >90% reduction in viability of leukemia cells with 50nM Dasatinib, however only ∼50% cell death at a much higher concentration of Nilotinib (1000nM). These data indicates that Dasatinib can efficiently kill TCF3-PBX1 leukemia cells by inhibiting pre-BCR and BCL6. ConclusionsOur study identified TCF3-PBX1 B-ALL as a unique form of ALL which has an unusual high activity of pre-BCR signaling. BCL6 was upregulated by pre-BCR, which we showed was required for proliferation and survival of TCF3-PBX1 ALL cells. Targeting pre-BCR and BCL6 using Dasatinib and specific BCL6 inhibitors (RI-BPI or PU-H71) induced apoptosis of primary TCF3-PBX1 B-ALL cells in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

RAG-Mediated Recombination Is The Predominant Driver Of Oncogenic Rearrangement In ETV6-RUNX1 Acute Lymphoblastic Leukemia

▪Approximately 25% of pediatric B-cell precursor ALL are characterized by the ETV6-RUNX1 fusion and are associated with a favorable prognosis. Monozygotic twin studies with concordant ALL and ‘backtracking' studies using archived neonatal blood spots established that ETV6-RUNX1 is an initiating event arising prenatally in a committed B-cell progenitor. However, the fusion gene is not sufficient on its own to cause overt leukemia and a number of subsequent studies have now provided strong evidence that additional mutations are essential for the clinical development of ALL.To obtain a detailed understanding of genetic events that drive this subtype of ALL, we carried out extensive genomic analysis in patients with ETV6-RUNX1 ALL. A total of 57 cases with diagnostic (leukemic) cell DNA paired with matched, remission samples as a source of constitutive DNA were used for exome sequencing (n=56) and low-depth wholegenome sequencing for structural variation (SV) analysis (n=51). Integrative analysis of exome and wholegenome data was performed and data were evaluated for recurrent gene mutations, copy number alterations and genomic rearrangements. Signatures of somatic mutation and structural variation were studied for insights into operative mutational processes and exposures that may drive ETV6-RUNX1pathogenesis.Wholegenome profiling identified 524 somatic SVs (average:11, range 0-49) including 34 tandem duplications, 66 inversions, 106 intrachromosomal translocations and 317 deletions. Resolution of the breakpoint junction to the base-pair level was possible for 354 SVs and in 40% of these breakpoints, we identified a significant enrichment of V(D)J recombination signal sequences or motifs that closely approximate these (E-value=3.8x10-116). Correlation with chromatin marks reveals a ten-fold genomewide enrichment of SVs (p<2.2x10-16) at promoters and enhancers of genes actively transcribed in early B-lineage development. Overall, 83% of resolved rearrangements mapping to promoters and 76% of rearrangements mapping to enhancer regions were in close proximity to a V(D)J recombination sequence motif as opposed to an average of 29% of the rearrangements in other chromatin states. These findings suggest that RAG-mediated genomic rearrangement is the dominant mutational process driving SV in ETV6-RUNX1 ALL. Single-cell tracking of genomic rearrangements bearing the hallmarks of aberrant V(D)J targeting in two patients, shows that these can occur multiple times and in distinct clones. This suggests that this mechanism is not restricted to one founder cell but is rather continuous during leukemic evolution and likely to contribute to the branching clonal phylogenies observed in ETV6-RUNX1ALL at presentation.Exome sequencing analysis confirmed 775 somatic substitutions and 16 indels across 715 protein coding genes. Oncogenic mutations in KRAS, NRAS, CTCF, DAXX, EZH2, and KDM6A are described as well as components of the cohesion complex including STAG2, SMC5 and SMC1A. Integrative analysis of whole-genome and exome sequencing data identified ATF7IP, MGA and STAG2 to be recurrently affected by both deletions or inactivating mutations and highlights their role as novel tumor suppressors in ETV6-RUNX1 ALL.Sequence context analyses on the acquired point mutations identifies two distinct mutational signatures that are operative in ETV6-RUNX1 ALL, signature A characterized by C>T transitions at CpGs and signature B characterized by C>G and C>T at TpCs, contributing 56% and 32% of all substitutions respectively. Signature B is consistent with the reported preference of the APOBEC family of enzymes and is often observed in patients presenting with a hypermutator phenotype and dense clustering of mutations.We report the identification of a novel spectrum of somatic mutations in ETV6-RUNX1 ALL and present the first detailed characterization of the genomic landscape of this ALL subtype. Our analysis shows that the critical secondary events to the ETV6-RUNX1 fusion gene formation are genomic rearrangements mostly driven by aberrant V(D)J recombination events targeting of actively transcribed genes during early B-Cell development. This represents a unique mechanism of genomic instability in cancer, that specifically targets the very genes required for normal B-Cell development and survival, which may in part explain the positive therapeutic response in this subtype of ALL. Disclosures:No relevant conflicts of interest to declare.

Pcft Is Silenced By DNA Methylation In Pediatric Acute Lymphoblastic Leukemia Resulting In Decreased Methotrexate Uptake

IntroductionAcute lymphoblastic leukemia (ALL) remains the most common cancer diagnosis in children, accounting for 21% of all pediatric cancers from birth to 19 years. Antifolate therapy, specifically treatment with methotrexate, is central in ALL therapy. Genetic polymorphisms, such as those for MTHFR and SLC01B1 can contribute to how methotrexate is metabolized and can alter side effect profiles, but they do not fully explain why transformed cells may become resistant to methotrexate. Epigenetic abnormalities, including dysregulation of DNA methylation, is frequent in all types of cancers including ALL, often leading to silencing of tumor suppressor genes. Epigenetic silencing could potentially influence a cell's sensitivity to antifolate therapy.PCFT is a low pH folate transporter and the regulation of PCFT gene expression includes DNA methylation. Previous studies have demonstrated that treatment with a hypomethylating agent results in reexpression of the gene. PCFT methylation, however, has not yet been studied in primary cancer tissues. Decreased or absent levels of PCFT could result in decreased methotrexate uptake and provide a mechanism of resistance. MethodsGenomic DNA and RNA were isolated from primary pediatric ALL samples as well as ALL cell lines. Methylation status was evaluated using methylation specific PCR (MSP). Expression was measured using qRT-PCR. Cell lines were treated with the hypomethylating agent 5-Aza-2-deoxycytidine (5-Aza-2dC). After treatment with 5-Aza-2dC, RNA was isolated for expression. Cells were also evaluated for methotrexate entry into the cell using fluorescein-tagged methotrexate. ResultsWe found that 100% of ALL cell lines (n=5) were methylated at PCFT. In primary patient samples, among an initial cohort, 23/33 samples (70%) had methylation at PCFT by MSP. By subtype of ALL, methylation of PCFT was found in 14/21 (67%) of B-cell ALL and 9/12 (75%) of T-cell ALL. This cohort did not have RNA available or outcome data. In a second pediatric ALL cohort of newly diagnosed patients, 23/40 (57%) were methylated at the PCFT promoter. Among these samples, 19/33 B-cell ALL, and 4/7 T-cell ALL were methylated at PCFT. The mean diagnostic age of the patients with PCFT promoter methylation was lower (7.13 years) than those that were unmethylated (8.71 years). Within this cohort with between 4.5 and 15 years of time elapsed since diagnosis, only 3 patients experienced recurrent or refractory disease. Of these patients, 1 had methylation of PCFT.Expression analysis by qRT-PCR demonstrated that 2 cell lines had no expression of PCFT (Tanoue and Nalm6) while 1 cell line expressed low level PCFT despite having promoter methylation (HB 11;19). Among the primary ALL samples, the unmethylated group had a higher mean expression of PCFT (1/ΔCt = 0.068) than the methylated group (1/ΔCt = 0.0635; p=0.045), suggesting that methylation of PCFT leads to decreased expression. Treatment of the ALL cell lines with 5-Aza-2dC did induces expression of PCFT in Nalm6 and Tanoue, but did not increase expression in HB 11;19 where expression was already present. After treatment with 5-Aza-2dC, Nalm6 and Tanoue also increased the intake of methotrexate by 2.25 and 1.7 times respectively, while in HB 11;19, the methotrexate intake decreased by a factor of 0.73. ConclusionsPCFT is frequently methylated in both B-cell and T-cell pediatric ALL. DNA methylation was associated with decreased expression in primary samples. Hypomethylating agents can reverse this gene silencing resulting in higher methotrexate entry into the cell, providing a potential strategy for overcoming methotrexate resistance. Disclosures:No relevant conflicts of interest to declare.

An Optimised In Vivo Modelling System For Adult Acute Lympoblastic Leukaemia (ALL) Enables Sensitive Detection Of Leukaemia Initiating Cells (LIC) and Drug Resistant Clones

Although humanised models of ALL are well described they are largely based on paediatric disease. The distinct nature of ALL in adults - evidenced by inferior outcome and different genetic landscape - points to a need for targeted study of this disease. Key to this is development of in vivo models of adult ALL. In this study we sought to establish the optimal modelling system for adult pre-B ALL using NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches. Our aims were to define the conditions for sensitive assaying of adult ALL LIC and low frequency clones.Pre-conditioning irradiation has been shown to negatively influence pre-B ALL engraftment. Studies of paediatric ALL suggest this may be due to reduced homing of ALL cells to the bone marrow (BM) resulting from aberrant CXCR4 receptor desensitisation induced by SDF-1 which is upregulated after irradiation. To investigate the impact of irradiation on engraftment of adult ALL, 5 - 10 million viable cells were injected intravenously (IV) into 6-8 week mice either unconditioned or (+24 hrs) following irradiation (2.5Gy). After 6 months, BM leukaemic engraftment (>0.1% hCD45+/19+) was assessed. Engraftment occurred in only 2/17 (11.8%) samples from non-irradiated recipients but in 11/14 samples (78.6%) after pre-conditioning irradiation (p = 0.0003). Comparison of 8 samples assayed by both models demonstrated that pre-conditioning irradiation was a pre-requisite for successful engraftment in 7/8 cases. Contrasting this overall finding was t(4;11) ALL where engraftment was higher in non-irradiated models (2/3).To determine homing capacity in adult ALL, SDF-1 (0, 10ng, 50ng, 125ng + 1ug) induced directional migration was investigated. A dose dependant response was observed with maximal migration (37±15 fold) of adult pre-B ALL cells at high (125ng/ul) concentrations of SDF-1 (n=5, non t(4;11) ALL). Hence, in contrast to paediatric ALL, adult ALL tumours do not appear to exhibit significant CXCR4 desensitisation to high levels of SDF-1 which may explain the positive effect of irradiation on adult ALL engraftment. Notably, t(4;11) positive tumours were relatively insensitive to SDF-1 induced migration (max fold change ∼2.5). CXCR4 cell surface receptor analysis revealed a trend towards low expression in t(4;11) cases (8.7±3.3% blasts) vs non t(4;11) (41 ±12% blasts) which may account for the differential SDF-1 induced in vitro migration patterns.Further modifications to the NSG xenotransplantation system were investigated: direct intra bone marrow (IBM) vs IV injection, 2.5Gy vs 2.0Gy and IBM +/- 2.5Gy irradiation. An IBM injection route, 2.5Gy irradiation and IBM + irradiation were all associated with an overall faster engraftment kinetic and time to disease manifestation. However, overall engraftment potential was not affected by these factors. Hence, the optimal xenotransplantation assay for adult ALL was defined as IBM delivery in 2.5Gy irradiated mice. This system enabled quantification of at least 1:1000 adult ALL LICs as assessed by limiting dilution transplantation (cell dose:103-106 ).Finally, we used the optimised xenotransplantation system to study 9 MRD positive ALL BM samples (8.33 x 10-2 – 2.64-4 positivity by BCR/ABL or I/g TCR based PCR quantification). MRD positive BM samples (unfractionated) were injected at a cell dose of 8.7 x104 – 6 x106/mouse and, strikingly, in the absence of cellular enrichment, MRD induced leukaemic engraftment (83.6%) was achieved in 1/9 samples after an extended modelling phase of 7 months and importantly gave rise to overt leukemia associated morbidity. Hence we establish proof of principle, that MRD + ALL can be modelled in vivo.In conclusion we have established the parameters of a functional system for in vivo study of adult ALL. Our findings highlight the key role of irradiation in the homing of adult ALL cells and also in their retention and repopulation, possibly mediated by SDF-1/CXCR4 interaction. Importantly, we found distinct in vivo growth requirements not only between adult ALL subtypes but also compared to paediatric disease underlining the importance of age/leukaemia specific investigation. Our optimised system should facilitate the study of LIC and pre-clinical disease modelling in adult ALL. Crucially we establish a strategy for assaying arguably the most informative populations (MRD) for understanding disease resistance and evolution of relapse. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Integrated Genomic and Mutational Profiling Of Adolescent and Young Adult ALL Identifies a High Frequency Of BCR-ABL1-Like ALL with Very Poor Outcome

The genetic basis underlying inferior outcome of adolescent and young adult acute lymphoblastic leukemia (AYA ALL) as compared to childhood cases is largely unknown. To comprehensively characterize the genetic landscape of AYA ALL we studied 423 adolescent (16-21 yrs; median 17.7±1.3 yrs) and 250 young adult (21-39 yrs; median 28.3±7.0 yrs) samples from the Children's Oncology Group high-risk trial AALL0232, St Jude Children's Research Hospital Total XV and XVI, Eastern Cooperative Oncology Group E2993, MD Anderson Cancer Center and the Alliance - CALGB trials. Single nucleotide polymorphism (SNP) microarray analysis and gene expression profiling were performed to identify copy number alterations and distinct genetic subgroups. Samples were also sub classified using hierarchical clustering, ROSE outlier and PAM analysis of gene expression profiling data. Sequence mutation analysis was performed on candidate genes known to be mutated in pediatric ALL (including IKZF1, PAX5, JAK1/2, NRAS, KRAS, FLT3, IL7R, SH2B3, TP53 and CREBBP), and mRNA-seq was performed on selected BCR-ABL1-like cases (n=41).The genetic subgroups were divided into ETV6-RUNX1, TCF3-PBX1, hyperdiploid (>50 chromosomes), MLL rearrangements, BCR-ABL1, BCR-ABL1-like, ERG and other (cases with no known lesions). As expected, ETV6-RUNX1 and hyperdiploid ALL were less frequent in adolescents (4% and 11%, respectively) and adults (2% for both) than in childhood ALL (<16 years; 25% for both). In contrast, the frequency of BCR-ABL1-like ALL, a recently described subgroup in 10-15% of pediatric ALL associated with kinase-activating lesions and a poor outcome, was very frequent and increased with age (21% in adolescent, 25% in young adults), similar to cases with the classic BCR-ABL1 translocation (6% in adolescent, 22% in young adults).Notably, BCR-ABL1 and BCR-ABL1-like ALL patients presented with higher white blood counts at diagnosis compared to non BCR-ABL1-like ALL patients in both adolescents (117.6 and 76.8 vs 21.9 x109/L, p<0001), and young adults (72.6 and 94.1 vs 17.6 x109/L, p<0001). BCR-ABL1-like ALL patients were also more likely to be male compared to non BCR-ABL1-like ALL patients, with 74% vs 62% in adolescents (p<0.05; Fisher's exact test), and 81% vs 63% in young adults (p=0.07; Fisher's exact test). The outcome of BCR-ABL1 and BCR-ABL1-like ALL was markedly inferior to other ALL subtypes, with 5-year event free survival (EFS) rates of 53.7+18.3 and 40.0+7.1 vs 85.0±3.3 (p<0.0001) in adolescent cases, and 23.2±9.1 and 16.1±8.5 vs 57.9±8.0 (p=0.006) for young adults (Figure 1). IKZF1 alterations, a marker of poor outcome in pediatric ALL, were enriched in BCR-ABL1 and BCR-ABL1-like ALL cases (70% and 77%, respectively) compared to non BCR-ABL1-like patients (26%). Regardless of genetic subtype, the presence of an IKZF1 alteration correlated with inferior 5 year EFS in adolescent (60.3±6.0 vs 77.4±4.1; p=0.0015) and young adults (25.7±7.0 vs 52.7±6.4; p=0.0011). [Display omitted] We then sought to characterize the alterations activating kinase signaling in AYA BCR-ABL1-like ALL cases. As observed in pediatric ALL, approximately 55% of these cases harbored CRLF2 rearrangements. Using mRNA-seq we identified a variety of additional rearrangements involving the tyrosine kinase or cytokine receptor genes ABL1, ABL2, CSF1R, JAK2, EPOR or PDGFRB, with a marked enrichment of fusions involving JAK2 (6 different fusions in 9/20 cases sequenced), thus providing a rationale for the investigation of targeted therapies directed against these alterations. Collectively, the kinase-activating BCR-ABL1 and BCR-ABL1-like subtypes are associated with poor outcome and make up ∼25% of adolescent and ∼50% of young adult ALL patients. The identification of these patients at diagnosis will provide an opportunity to incorporate tyrosine kinase inhibitor treatment to current chemotherapeutic regimens, and significantly improve the treatment outcome for AYA ALL. Disclosures:Hunger:Bristol Myers Squibb: Consultancy.

IKZF1 deletions Are Detected In 40.3% Of 270 Adult B-Precursor-ALL and Are Independently Associated With BCR-ABL1 Positivity and Inferior Outcome

▪ IntroductionTotal or intragenic deletions of or within the IKZF1 (Ikaros) gene on chromosome 7 have been shown to be frequent in B-precursor-ALL (B-ALL), highly associated with BCR-ABL1 positive (BCR-ABL1pos) B-ALL (Mullighan et al., Nature 2008), but also present in BCR-ABL1 negative (BCR-ABL1neg) B-ALL (Mullighan et al., N Engl J Med 2009). IKZF1 deletion pattern is known to be heterogenous and deletions involving exon 4, 7 or 8 are likely to have the same impact as whole gene deletions. To analyse intragenic IKZF1 deletions, we used a multiplex-PCR approach, which represents the standard detection method. In addition, we used multiplex ligation-dependent probe amplification (MLPA), which allows the detection of whole IKZF1 deletions. AimWe aimed at characterization of IKZF1 deletions pattern and correlation to clinical features in a cohort of 270 adult B-ALL cases. Patients and MethodsIKZF1 deletion status was analyzed in blood or bone marrow samples from 270 adult B-ALL cases (subtypes as diagnosed by immunophenotyping: c-ALL: n=197; Pro-B ALL: n=51; Pre-B ALL: n=5). Additionally, 17 mature B-ALL cases were analyzed. The cohort consisted of 137 females and 133 males, median age was 58.3 years (range: 18.1-91.4 years). The cohort was classified into seven subgroups according to the following cytogenetics: 1) t(9;22)(q34;q11) (n=97), 2) 11q23/MLL rearrangements (n=24), 3) MYC rearrangements (n=14), 4) hypodiploidy (n=21), 5) hyperdiploidy (n=32), 6) normal karyotype (n=38), 7) other cytogenetic aberrations (n=42). In one case no cytogenetic data was available. In all 270 cases intragenic IKZF1 deletions were investigated by breakpoint-specific fluorescent multiplex PCR (according to Caye et al., Hematologica 2012). In 206 cases we additionally used MLPA (P335 SALSA MLPA kit IKZF1, MCR Holland, The Netherlands) to identify whole IKZF1 deletions not detectable by multiplex-PCR. ResultsIn total, 132 IKZF1 alterations were identified in 109/270 cases (40.3%). With regard to ALL subtypes in c-ALL 47.7% (94/197) IKZF1 mutations were detected, in Pro-B ALL 23.5% (12/51) and in three of five Pre-B ALL cases. No IKZF1 deletions were detected in mature B-ALL and thus were mutually exclusive with MYC rearrangements. There was no significant difference in age, sex, leukocyte count, hemoglobin level or platelet count between patients with or without IKZF1 deletions, respectively. With regard to immunophenotype cases with IKZF1 deletions had a stronger expression of CD13 (38±28% vs. 27±25% positive cells; p=0.004), CD33 (24±23% vs. 17±21%; p=0.008), CD34 (72±25% vs. 43±35%; p<0.001%) and TdT (61±28% vs. 43±32%; p<0.001).In 86/109 cases (78.9%) one (monoallelic) intragenic IKZF1 deletion was detected by multiplex-PCR and MLPA. In detail, 38 patients (34.9%) showed deletions of exon 4-7, 15 cases (13.7%) deletions of exon 2-7, 5 cases (4.6%) deletions of exon 4-8 and 3 cases (1.8%) deletions of exon 2-8. In one case, a deletion of IKZF1 exons 2 and 3 was detected by MLPA. 23/109 cases (21.1%) showed two (biallelic) IKZF1 deletions. Whole IKZF1 gene deletions were detected by MLPA in 24/109 cases (22.0%) and were associated with cytogenetic abnormalities of the short arm of chromosome 7 in 21/40 cases (52.5%) including: monosomy 7 (n=14), i(7)(q10) (n=2), dicentric chromosomes 7 (n=1), and unbalanced translocations involving chromosome 7 (n=4). Four of 24 cases with MLL-rearrangements harbored IKZF1 deletions, three of these being whole IKZF1 gene deletions due to monosomy 7.IKZF1 deletions were highly associated with BCR-ABL1 positivity: 72/97 BCR-ABL1pos (74.2%) versus 37/172 BCR-ABL1neg cases (21.5%) (p<0.001). 61/85 cases (71.8%) with intragenic IKZF1 deletions were BCR-ABL1pos compared to 11/24 (45.8%) cases with whole IKZF1 deletions (p=0.018). In 97 BCR-ABL1pos cases, the additional presence of IKZF1 deletions was correlated to inferior survival (p=0.070). Conclusions1) IKZF1 deletions were detected in 40.3% in adult B-ALL by using muliplex-PCR and MLPA. 2) Whole but also intragenic IKZF1 deletions are highly associated with BCR-ABL1 in adult B-ALL. 3) As 22% of IKZF1 deletions were whole gene deletions not detectable by standard multiplex-PCR, at least two molecular methods (multiplex-PCR and MLPA) are required to fully detect the whole spectrum of IKZF1 deletion patterns in adult B-ALL. Disclosures:Fasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ulke:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Acute Lymphoblastic Leukemia Is a Bcl-2 Dependent Disease: Proteomic Profiling and Pre-Clinical Efficacy Of a Selective Bcl-2 Antagonist ABT-199

The expression of Bcl-2 family proteins is perturbed in multiple types of cancers, including leukemias, and is associated with disease progression and resistance to chemotherapy. ABT-199 (GDC-0199) is a new BH3 mimetic that was developed to specifically target Bcl-2 while sparing Bcl-XL, hence avoiding thrombocytopenia intrinsic to 1st generation BH3 mimetics like ABT-737 (Souers et al., Nat Med, 2013). In this study, we report proteomic profiling of Bcl-2 family members in a large series of ALL patients (pts) and pre-clinical activity of ABT-199.Expression of 20 pro- and anti-apoptotic proteins was studied in 186 newly diagnosed ALL using reverse phase protein arrays (RPPA). Supervised clustering demonstrated distinct differences in 11 proteins in ALL with different cytogenetic and FAB characteristics (Fig. 1, p<0.005, false discovery rate <0.2%). Among these, pts with Burkitt's leukemia/lymphoma (n=9) expressed low levels of Bcl-2 and Bax while maintaining high expression of Bim, caspases and PARP. In contrast, t(4;11) pts expressed higher levels of Bcl-2, Bax and Bim. No significant differences in Bcl-XL or Mcl-1 levels were found in different ALL subtypes. [Display omitted] The potential of ABT-199 to disrupt interactions between Bcl-2 and different pro-apoptotic proteins was studied using Bimolecular Fluorescence Complementation (BiFC, J Biol Chem 288:4935, 2013). The coding sequences for human Bcl-2, Bim, Bak, Bax and Noxa were subcloned into BiFC plasmids containing Venus fragments and transfected into HeLa cells. Approximately 60-70% of transfected cells were positive for Venus fluorescence due to association between Bcl-2 and Bim, Noxa, Bax or Bak. ABT-199 (2.5 µM, 24 hrs) significantly reduced Venus signal, indicating an inhibition of the interactions of Bcl-2 with these proteins, most potently with the multidomain proteins Bax and Bak (95%±18% and 85%±15% inhibition, respectively). ABT-199 rapidly induced apoptotic cell death in ALL cell lines and in primary ALL samples. Pre-B ALL cells (Nalm-6, REH, SEMK2 and RS4;11) were sensitive to ABT-199 and ABT-737 (IC50 0.007-1.4µM (199) and 0.035-0.7µM (737)). Notably, ABT-199 was more cytotoxic than ABT-737 against MLL-rearranged SEMK2 and RS4;11 cells, consistent with the notion of the greater Bcl-2 dependency of these cells. Lentiviral silencing of Bcl-XL sensitized REH cells to apoptosis by ABT-199 and ABT-737. T-ALL cells (PF-382, MOLT-4, P-12) expressed lower levels of Bcl-2 and were uniformly less sensitive to ABT-199 compared to ABT-737 (IC50 3.7±1.1µM vs 0.7±0.3µM, p=0.01). Burkitt's lymphoma cells Ramos and Raji had low Bcl-2 and high Mcl-1 expression, and were resistant to both agents (IC50>4µM).Next, the cytotoxic activity of ABT-199 was tested against a panel of 12 genetically diverse primary ALL samples, including 6 from pts with relapsed or refractory disease. Ten out of twelve samples (83%) were exquisitely sensitive to both agents, with IC50 values of 0.0001-0.14µM for ABT-199 and 0.0004-0.3µM for ABT-737. One of the four Ph+ samples was resistant to both agents, and one of the two T-ALL was less sensitive to ABT-199 compared to ABT-737. Two samples with t(4;11) were highly sensitive to ABT-199. All primary ALL samples tested (n=7) expressed high levels of Bcl-2, and no significant correlation between sensitivity and expression of Bcl-2 family members was found. Importantly, three human-derived xenografts from pediatric pre-B-ALL samples (1345, 1809, 0398) were very sensitive to ABT-199 (IC50 3nM, 0.1nM and 2.3nM, respectively). Finally, anti-leukemia activity of ABT-199 was tested in MLL-rearranged patient-derived xenograft NSG mice. Treatment with ABT-199 at 100mg/kg/d by oral gavage days 13-23 significantly reduced leukemia tumor burden as determined by bioluminescence imaging (average 70% reduction in BLI signal in 4 ABT-treated mice compared to 4 control animals at 9 weeks, p=0.03).In summary, proteomic profiling and patterns of sensitivity to Bcl-2 inhibition indicate that ALL, with exception of Burkitt's lymphoma, represents a Bcl-2 dependent disease. These results provide strong rationale for introducing ABT-199, which recently showed impressive efficacy in CLL trials, into the clinical armamentarium of ALL therapy. Disclosures:Konopleva:AbbVie, Inc: Research Funding. Leverson:AbbVie, Inc.: Employment, Equity Ownership.

Implication of IRF4 Aberrant Gene Expression in the Acute Leukemias of Childhood

The most frequent targets of genetic alterations in human leukemias are transcription factor genes with essential functions in normal blood cell development. The Interferon Regulatory Factor 4 (IRF4) gene encodes a transcription factor important for key developmental stages of hematopoiesis, with known oncogenic implications in multiple myeloma, adult leukemias and lymphomas. Very few studies have reported an association of IRF4 with childhood malignancy, whereas high transcript levels have been observed in the more mature immunophenotype of ALL. Our aim was to investigate the expression levels of IRF4 in the diagnostic samples of pediatric leukemias and compare them to those of healthy controls, in order to determine aberrant gene expression and whether it extends to leukemic subtypes other than the relatively mature ALL subpopulation. Quantitative real-time RT-PCR methodology was used to investigate IRF4 expression in 58 children with acute leukemias, 4 leukemic cell lines and 20 healthy children. We show that aberrant IRF4 gene expression is implicated in a variety of leukemic subtypes; higher transcript levels appear in the more immature B-common ALL subtype and in T-cell than in B-cell leukemias, with the highest expression levels appearing in the AML group. Interestingly, we show that childhood leukemia, irrespective of subtype or cell maturation stage, is characterised by a minimum of approximately twice the amount of IRF4 gene expression encountered in healthy children. A statistically significant correlation also appeared to exist between high IRF4 expression and relapse. Our results show that ectopic expression of IRF4 follows the reverse expression pattern of what is encountered in normal B-cell development and that there might be a dose-dependency of childhood leukemia for aberrantly expressed IRF4, a characteristic that could be explored therapeutically. It is also suggested that high IRF4 expression might be used as an additional prognostic marker of relapse at diagnosis.

Integration of High-Resolution Methylome and Transcriptome Analyses to Dissect Epigenomic Changes in Childhood Acute Lymphoblastic Leukemia

B-cell precursor acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer. Although the genetic determinants underlying disease onset remain unclear, epigenetic modifications including DNA methylation are suggested to contribute significantly to leukemogenesis. Using the Illumina 450K array, we assessed DNA methylation in matched tumor-normal samples of 46 childhood patients with pre-B ALL, extending single CpG-site resolution analysis of the pre-B ALL methylome beyond CpG-islands (CGI). Unsupervised hierarchical clustering of CpG-site neighborhood, gene, or microRNA (miRNA) gene-associated methylation levels separated the tumor cohort according to major pre-B ALL subtypes, and methylation in CGIs, CGI shores, and in regions around the transcription start site was found to significantly correlate with transcript expression. Focusing on samples carrying the t(12;21) ETV6-RUNX1 fusion, we identified 119 subtype-specific high-confidence marker CpG-loci. Pathway analyses linked the CpG-loci-associated genes with hematopoiesis and cancer. Further integration with whole-transcriptome data showed the effects of methylation on expression of 17 potential drivers of leukemogenesis. Independent validation of array methylation and sequencing-derived transcript expression with Sequenom Epityper technology and real-time quantitative reverse transcriptase PCR, respectively, indicates more than 80% empirical accuracy of our genome-wide findings. In summary, genome-wide DNA methylation profiling enabled us to separate pre-B ALL according to major subtypes, to map epigenetic biomarkers specific for the t(12;21) subtype, and through a combined methylome and transcriptome approach to identify downstream effects on candidate drivers of leukemogenesis.

Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies.

BackgroundDespite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies.Methodology and Principal FindingsUsing flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues.Conclusions and SignificanceCollectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.