ALBERT

Description: Introduction: Ca2+ release-activated Ca2+ (CRAC) channels and their activators stromal interaction molecule (STIM) 1 and 2 are the main regulators of calcium entry in T Lymphocytes through a process known as store-operated Ca2+ entry (SOCE). SOCE results in the activation of calcineurin and other downstream signals with important effects on lymphocyte function. Notch-1 is a protein that is essential for T lymphocyte development. Activating mutations of Notch-1 occurs in about 60% of T-cell acute lymphoblastic leukemia (T-ALL). Introduction of constitutively active forms of Notch-1 in hematopoietic stem cells (HSC) induces T-ALL in mice, providing a useful animal model for the study of leukemia. Methods: To study the role of CRAC channels in T-ALL, we used a mouse model in which c-kit+ HSC from wild-type (WT) and STIM1/STIM2-deficient mice (DKO) were retrovirally transduced with the intracellular Notch-1 domain (ICN1). Transduced HSC were injected into lethally irradiated C57BL/6 mice. Following leukemia development, mice were analyzed for survival and cellular and molecular activity of leukemic cells using various techniques including histology, flow cytometry, RT-PCR and gene array expression analysis. In addition, we used the human T-ALL cell line CEM, in which we introduced a dominant negative form of the CRAC channel subunit ORAI1 (ORAI1-DN) that abolishes CRAC channel function and SOCE, for coculture with the human bone marrow stromal cell line HS5. Results: Mice injected with wild-type HSC transduced with ICN1 succumbed from T-ALL characterized by the presence of CD4+ CD8+ leukemic T cell blasts in the blood, bone marrow and infiltrating organs within 3 to 4 weeks after transfer of HSC. By contrast, mice that had received ICN1 transduced STIM1/2 deficient HSC lived approximately twice as long. The survival benefit was not due to differences in leukemic cell numbers or in proliferation and apoptosis of leukemic cells. Histologies of the bone marrow and spleen of WT leukemic mice showed necrotic lesions, pronounced neutrophil infiltration, the presence of histiocytes engulfing red blood cells (RBC) indicative of severe inflammation. No signs of necrosis and inflammation were present in DKO leukemic mice. Paralleling the inflammation and destruction of the bone marrow environment, WT leukemic mice showed greatly diminished presence of erythroid precursors (EP) in the bone marrow whereas EP frequencies in DKO leukemic mice were similar to those in non-leukemic mice. In line with findings in mice, we observed that human leukemic CEM T cells reduced the viability of HS5 stromal cells in a contact-dependent manner. This cytotoxic effect of CEM cells depended on CRAC channel function as CEM cells transduced with ORAI1-DN had little effect on HS5 viability. Conclusion: These results suggest that CRAC channels are important for the function of T-ALL cells and their effects on the organs they infiltrate, most notably the bone marrow. Inhibition of CRAC channel function prolongs survival of mice with T-ALL potentially by attenuating the cytotoxic effects of leukemic T cells on their environment and on hematopoiesis. Further studies are underway to understand the mechanisms by which CRAC channels regulate leukemic T cell function. Disclosures Feske: Calcimedica: Consultancy, Equity Ownership, Honoraria, Patents & Royalties: CRAC Channel Inibitors.

Description: Background: The CALGB 10603 RATIFY trial demonstrated that midostaurin, a multi-targeted small molecule FLT3 inhibitor, when given in combination with standard 3+7 induction consisting of daunorubicin 60mg/m2 for 3 doses and cytarabine 200mg/m2/day for 7 days significantly prolonged overall survival (OS) compared to placebo plus standard induction in FLT3 positive acute myeloid leukemia (AML) patients. The safety and efficacy of the other anthracycline commonly utilized in standard AML induction, idarubicin, has not been evaluated to date. Methods: A single-center retrospective review from May 2017 to July 2018 was performed. Patients were included if they had a diagnosis of FLT3 positive AML and received induction chemotherapy with idarubicin. The primary outcome was incidence of adverse effects attributed to midostaurin. Grading of adverse effects was in accordance with the Common Terminology and Criteria of Adverse Effects (CTCAE) version 5. Additional outcomes included OS, relapse-free survival (RFS), similar as in the Ratify trial, as well as detection of FLT3 on subsequent bone marrow biopsies. Results: Ten patients were included. All patients received induction therapy with idarubicin 12mg/m2 for 3 doses and cytarabine 100mg/m2/day for 7 days. Median age was 53 years (range: 33 to 66) and 6/10 were male. Eight of 10 patients exhibited internal tandem duplication (ITD) on diagnosis; two had FLT3 tyrosine kinase domain (TKD) D835. Eight patients had diploid cytogenetics; the other two patients had core-binding factor AML. Midostaurin was initiated on day 8 of induction in all but 2 patients, who started on day 11 and 12, respectively. Nine of ten patients completed all 28 planned doses of midostaurin. All patients received antifungal prophylaxis with micafungin throughout the course of midostaurin. The median time from day 1 of induction to neutrophil (〉500/µl) and platelet (〉100,000/µl) recovery was 23 days and 26 days, respectively. Granulocyte colony stimulating factor (G-CSF) was initiated for all patients after day 14 bone marrow biopsy, as per institutional procedure. Four of 10 patients experienced an adverse event attributed to midostaurin. Maculopapular rash was observed in 3 patients a median of 5 days after midostaurin initiation: 2 of 3 patients had a grade 2 rash and continued therapy with topical steroids; one patient had a grade 3 rash and discontinued midostaurin after 17 of 28 planned doses. A grade 1 drug fever was attributed to midostaurin in a fourth patient. Fevers persisted despite neutrophil recovery and subsequently dissipated after completion of the final midostaurin dose. Persistent FLT3 mutation was detected in 4/9 (1 not reported) day 14 bone marrow biopsies but was negative in 9/10 pts on day 28. The lone positive FLT3 result on day 28 occurred in a patient with refractory disease (〉5% blasts) necessitating salvage therapy. Notably, this patient only completed 17 of 28 planned doses. All other patients exhibited a complete response (CR) on day 28. The median follow-up time was 243 days (range: 57 to 394). Nine patients are alive at the time of reporting. Six patients proceeded to allogeneic transplantation -one death was attributed to transplant-related complications, occurring in the same patient needing salvage and reduced duration midostaurin. Two patients relapsed a median of 184 days after start of induction -both FLT3-ITD positive and neither having undergone allogeneic transplant prior to relapse. Conclusions: Midostaurin in combination with idarubicin-based induction 3+7 therapy in this first case series appears to be safe. While the incidence of rash was higher (30%) than reported in RATIFY, this only resulted in discontinuation of therapy in one patient. Although patient numbers are limited, 90% achieved a FLT3 negative CR after completion of induction therapy and six patients proceeded to allogeneic transplantation. A confounding variable includes the routine use of G-CSF, which likely contributed to the shorter duration from induction to neutrophil recovery observed compared to RATIFY (23 days vs 26 days). The influence of G-CSF use on outcome is uncertain, however represents an interesting observation. A randomized, prospective trial comparing midostaurin in combination with idarubicin versus daunorubicin at both 60mg/m2 and 90mg/m2 is warranted to establish the optimal anthracycline induction therapy for FLT3 mutated AML patients. Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Diefenbach:Denovo: Research Funding; Merck: Consultancy, Research Funding; Acerta: Research Funding; Incyte: Research Funding; Trillium: Research Funding; Millenium/Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy; Seattle Genetics: Consultancy, Research Funding.

Description: Background: Outcomes for children with acute lymphoblastic leukemia (ALL) have dramatically improved, but survival for patients who relapse remains poor. Mutations in genes encoding epigenetic modifiers are present in the majority of patients at relapse. In particular, activating mutations in NSD2 (MMSET, WHSC1), namely the glutamate to lysine substitution at amino acid 1099 (p.E1099K), are among the most common such mutations in epigenetic regulators. NSD2 converts histone 3 lysine 36 (H3K36) into its dimethylated form (H3K36me2) which in turn leads to stereotactic inhibition of EZH2 mediated H3K27me3. We and others have established that this leads to changes in chromatin state and gene expression. However, the pathways by which this leads to a clonal advantage remains elusive. Design/Method: We previously reported that overexpression of wild-type (WT) and p.E1099K mutant (EK) NSD2 in B-ALL cell lines led to unique cell context specific chromatin alterations and altered gene expression but did not lead to changes in proliferation or intrinsic drug resistance in vitro (Pierro et. al. Blood 2017 130:2474). We reasoned that these observations could be explained by the need for cooperating pathways that together with NSD2 EK lead to a clonal advantage. Thus we modulated expression of NSD2 using short hairpin RNAs (shRNAs) in the B-ALL cell line RS4;11 which harbors a heterozygous NSD2 EK mutation (NSD2 low). As a control, RS4;11 was also stably transduced with a non-targeting shRNA sequence (NSD2 high). Knockdown of NSD2 as well as decrease in H3K36me2 in NSD2 low lines was confirmed by Western Blot. Differences in gene regulation in NSD2 low cells were assessed by ChIPseq for CTCF, H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3, and the results were correlated with RNAseq data. This data was then compared to RNAseq and ChIPseq data from REH and 697 NSD2 WT and EK overexpression cell lines in an effort to identify candidate genes or pathways preferentially regulated by the NSD2 EK mutation. Results: NSD2 low cells displayed a distinct gene expression profile compared to NSD2 high with 301 upregulated and 573 downregulated genes (LFC 0.58, P = 0.05). When compared to gene expression data from our previously reported NSD2 overexpression cell lines, there was minimal overlap across cell lines with only 15 differentially expressed genes shared between RS4;11 NSD2 knockdown and REH EK overexpression cell lines and only 24 genes shared between RS4;11 NSD2 knockdown and 697 EK overexpression cell lines. Across all cell lines (RS4;11, REH and 697), only three genes (NSD2, SCN8 and PCNXL2) overlapped, all of which were upregulated in NSD2 high cell lines. Using less stringent criteria (LFC 0.26, P = 0.1), we observed greater overlap with 34 shared up and downregulated genes among lines. Of the shared genes, only ZNF521 which is overexpressed in NSD2 high cell lines, is known play a role in leukemogenesis. Moreover, RS4;11 pathway analysis revealed several biologically relevant pathways modulated by the NSD2 EK mutation such as Ras, integrin signaling, cholesterol/steroid biosynthesis, apoptosis and cell proliferation. Significant differences were also observed across epigenetic marks between RS 4;11 NSD2 high and low cells. In accordance with previously published data, we observed a global decrease in the H3K36me2 mark in RS4;11 NSD2 low lines. When aligned with changes in histone marks, among genes downregulated in NSD2 low cells there was a clear correlation with acquisition of the repressive H3K27me3 mark (and a decrease in the H3K9Ac mark). However among genes upregulated in NSD2 low cells we saw paradoxical increases in the H3K36me2 mark and decreases in the H3K27me3. Furthermore, gene expression was also influenced by marks not directly regulated by NSD2, namely H3K27ac and H3K9ac, indicating that local NSD2 mediated epigenetic changes are not the sole regulator of gene expression. Conclusion: The activating p.E1099K substitution in NSD2 leads to a distinct gene expression profile in B-ALL cell lines that is cell context dependent. Moreover, while there is significant overlap in the transcriptional profile between WT and EK overexpression, there are distinct differences possibly indicating novel properties of the pE1099K substitution beyond enzyme hyperactivation. Our findings also imply that NSD2 EK collaborates with other leukemia associated alterations that result in clonal selection. Disclosures No relevant conflicts of interest to declare.

Description: Background: The treatment paradigm of adult patients with acute lymphoblastic leukemia (ALL) is primarily derived from successful pediatric chemotherapy regimens. Pegasparagase (PEG) is a key component of pediatric therapy and is the backbone of cytotoxic ALL regimens. However, among the adult population the use of PEG has been limited by the difficulty in tolerating prolonged asparagine depletion. Hepatotoxicity is among the most common adverse events reported with the use of PEG, with grade 3/4 hepatotoxicity seen in 20% of young adults compared to 40-60% of older adults. Incorporating PEG into the treatment of ALL patients under 40 remains an accepted practice despite some studies that report up to 75% of patients have grade 3/4 adverse events as a result of asparagine depletion. In a study of 85 patients with ALL, 3-year overall survival (OS) was significantly different between patients older and younger than 35 (52% vs 83% p = 0.003). Whether this difference is due to PEG toxicity or to other factors remains to be determined. At NYU hospitals, PEG-containing protocols are frequently deployed to treat adult ALL. In our study, we sought to look at the difference in PEG toxicity and response rate (RR) in patients older and younger than 35 and whether these toxicities contributed to a delay in subsequent treatments and to a worse outcome. Methods: We conducted a retrospective chart review of patients older than 18 diagnosed with ALL or lymphoblastic lymphoma, who received at least 1 dose of PEG at our institution between 2014 and 2018. All patients received PEG as part of their first line treatment protocol. Our main objective was to compare the tolerability and toxicity profile of intravenous PEG in patients ≥35 years old versus