ALBERT

Description: Abstract 115 Extra copies of chromosome 21 (polysomy 21) is the most common somatic aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL), including 〉90% of cases with high hyperdiploidy. In addition, children with Down syndrome (DS) have a 20-fold increased risk of developing B-ALL, of which ∼60% harbor CRLF2 rearrangements. To examine these associations within genetically defined models, we investigated B-lineage phenotypes in Ts1Rhr mice, which harbor triplication of 31 genes syntenic with the DS critical region (DSCR) on human chr.21. Murine pro-B cell (B220+CD43+) development proceeds sequentially through “Hardy fractions” defined by cell surface phenotype: A (CD24−BP-1−), B (CD24+BP-1−) and then C (CD24+BP-1+). Compared with otherwise isogenic wild-type littermates, Ts1Rhr bone marrow harbored decreased percentages of Hardy fraction B and C cells, indicating that DSCR triplication is sufficient to disrupt the Hardy A-to-B transition. Of note, the same phenotype was reported in human DS fetal liver B-cells, which have a block between the pre-pro- and pro-B cell stages (analogous to Hardy A-to-B). To determine whether DSCR triplication affects B-cell proliferation in vitro, we analyzed colony formation and serial replating in methylcellulose cultures. Ts1Rhr bone marrow (B6/FVB background) formed 2–3-fold more B-cell colonies in early passages compared to bone marrow from wild-type littermates. While wild-type B-cells could not serially replate beyond 4 passages, Ts1Rhr B-cells displayed indefinite serial replating (〉10 passages). Ts1Rhr mice do not spontaneously develop leukemia, so we utilized two mouse models to determine whether DSCR triplication cooperates with leukemogenic oncogenes in vivo. First, we generated Eμ-CRLF2 F232C mice, which express the constitutively active CRLF2 mutant solely within B-cells. Like Ts1Rhr B-cells, (but not CRLF2 F232C B-cells) Ts1Rhr/CRLF2 F232C cells had indefinite serial replating potential. In contrast with Ts1Rhr B-cells, Ts1Rhr/CRLF2 F232C B-cells also engrafted into NOD.Scid.IL2Rγ−/− mice and caused fatal and serially transplantable B-ALL. Second, we retrovirally transduced BCR-ABL1 into unselected bone marrow from wild-type and Ts1Rhr mice and transplanted into irradiated wild-type recipients. Transplantation of transduced Ts1Rhr cells (106, 105, or 104) caused fatal B-ALL in recipient mice with shorter latency and increased penetrance compared to recipients of the same number of transduced wild-type cells. By Poisson calculation, the number of B-ALL initiating cells in transduced Ts1Rhr bone marrow was ∼4-fold higher than in wild-type animals (1:60 vs 1:244, P=0.0107). Strikingly, transplantation of individual Hardy A, B, and C fractions after sorting and BCR-ABL1 transduction demonstrated that the increased leukemia-initiating capacity almost completely resides in the Ts1Rhr Hardy B fraction; i.e., the same subset suppressed during Ts1Rhr B-cell differentiation. To define transcriptional determinants of these phenotypes, we performed RNAseq of Ts1Rhr and wild-type B cells in methylcellulose culture (n=3 biologic replicates per genotype). As expected, Ts1Rhr colonies had ∼1.5-fold higher RNA abundance of expressed DSCR genes. We defined a Ts1Rhr signature of the top 200 genes (false discovery rate (FDR) 2,300 signatures in the Molecular Signatures Database (MSigDB) C2 Chemical and Genetic Perturbations with the Ts1Rhr signature identified enrichment in multiple gene sets of polycomb repressor complex (PRC2) targets and H3K27 trimethylation. Most notably, SUZ12 targets within human embryonic stem cells were more highly expressed in Ts1Rhr cells (P=1.2×10−6, FDR=0.003) and the same SUZ12 signature was enriched in patients with DS-ALL compared to non-DS-ALL (P=0.007). In summary, DSCR triplication directly suppresses precursor B-cell differentiation and promotes B-cell transformation both in vitro and by cooperating with proliferative alterations such as CRLF2 activation and BCR-ABL1 in vivo. Pharmacologic modulation of H3K27me3 effectors may overcome the pro-leukemogenic effects of polysomy 21. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3627 Background: In pts with mutant FLT3 AML single agent studies with inhibitors of FLT3, such as midostaurin, yielded a high rate of peripheral blast reduction but complete and partial remissions were uncommon. One approach to overcome resistance is to combine a FLT3 inhibitor with an agent that down-regulates another leukemogenic pathway including those downstream to activated FLT3. Preclinical work has suggested that mTOR inhibitors, such as rapamycin or its analogs including everolimus, and the FLT3 inhibitor midostaurin synergistically kill mutant FLT3 dependent cell lines. Aims: To identify the maximum tolerated dose (MTD) of everolimus that can be given in combination with 50 mg orally twice daily midostaurin (established as well tolerated and capable of decreasing peripheral blast count [Fischer JCO 2010]), to determine toxicities, to observe anti-leukemic effects, to measure pharmacokinetics of each agent when administered in combination, and to observe pharmacodynamic effects including decrease in the phospho-FLT3 to total FLT3 ratio in blasts as evidence of enzyme inhibition. Methods: In this phase I study pts were treated with a combination of midostaurin at 50 mg po BID continuously beginning on day 2 and everolimus po administered on day 1 and continuously from day 8 onward. Dose escalation of everolimus was conducted according to standard 3 + 3 schema; the doses of everolimus explored were 5 mg QOD (dose level 1) and 5 mg QD (dose level 2). An additional 15 pts were enrolled at the MTD. Results: A total of 29 pts were enrolled (24 with relapsed and/or refractory AML, 4 with newly diagnosed AML considered inappropriate candidates for standard therapy, and 1 with relapsed CMML; 11 FLTwt, 14 FLT3-ITD positive, 3 FLT3 D835Y positive, 1 FLT3 D835H positive). The mean age was 66 (range 40–85) and ECOG performance statuses were 0 in 6 pts, 1 in 16 pts, and 2 in 7 pts. After 1 of 3 pts treated at the everolimus starting dose of 5 mg QOD experienced a DLT (grade 5 infection) the cohort was expanded to 6 pts; of the latter 3 pts enrolled 1 experienced a DLT of grade 3 hypoxia. The eligibility criteria were subsequently amended so as to be more stringent, including a DLCO requirement of 50% predicted. After an additional 3 evaluable pts tolerated the 5 mg QOD dose, we escalated to 5 mg QD; 3 of 4 pts treated at this dose level experienced DLT (grade 3 hypoxia, grade 3 mucositis, grade 5 soft-tissue infection), and subsequent pts were treated at the MTD, everolimus 5 mg QOD (25 pts in total received this dose). The most common drug-related toxicities at the MTD (any grade) were fatigue (40%), nausea (32%), hypertriglyceridemia (24%), vomiting (24%), hyperglycemia (20%), and elevated AST (20%). In addition to the pt who died in the first everolimus cohort, 5/25 pts discontinued due to drug-related grade 3 toxicity, including pneumonia (1), hypoxia (2), allergic reaction (1), and nausea (1). Grade 3 events that did not result in treatment discontinuation included thrombocytopenia (1), dyspnea (2), hyponatremia (1), hypokalemia (1), gum pain (1), abdominal pain (1), pneumonitis (1), and febrile neutropenia (2). One mutant FLT3 D835Y pt achieved CR and went on to allogeneic SCT. 3 pts (2 FLT3-ITD positive, 1 FLTwt) experienced a blast response (a ≥50% peripheral or bone marrow blast reduction) with 2 of these pts ultimately progressing and 1 discontinuing due to toxicity. Overall blast response or better was achieved in 4/29 pts (14%), 8 pts had stable disease, and 17 pts had progressive disease and/or received less than 15 days of therapy (6). In the 6 pts who received less than 15 days of therapy reasons for treatment termination included grade 3 (1) and grade 5 toxicity (2), progressive disease (2), and physician decision (1). The incidence of blast response or better in FLT3mut pts (4/18, 22%) was not clearly higher than that observed in other studies involving midostaurin administered at 50 mg po BID as a single agent. Pharmacokinetic and pharmacodynamic data are being analyzed, though at least one patient's blasts revealed a treatment-related decrease in the phospho FLT3/FLT3 ratio, suggesting enzyme inhibition. Conclusion: The combination of midostaurin (50 mg BID) and everolimus (5 mg QOD) is tolerable and has efficacy in AML, but it is unclear whether the addition of the rapamycin analog results in superior outcomes compared with midostaurin alone. Disclosures: Stone: Novartis: Consultancy, Research Funding. Off Label Use: evorlimus in AML. Griffin:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy.