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Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response. Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling. Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

Background: Despite improvements in outcomes for children with B- and T-cell acute lymphoblastic leukemia and lymphoblastic lymphoma (B-ALL, T-ALL, B-LLy and T-LLy), the outcome of patients with primary resistant or relapsed disease remains very poor, particularly for children with second or greater relapses and with T-cell disease. Deregulation of cell cycle machinery is essential for both the induction and progression of ALL (Sicinska et al. Cancer Cell 4:451-61, 2003). Additionally, targeting cell cycle regulators CDK4 and 6 efficiently suppresses ALL growth and disease progression in vivo (Sawei et al. Cancer Cell 22:452-65, 2012) and this effect is augmented by conventional chemotherapy. These data provided the rationale for investigating the CDK4/6 inhibitor palbociclib in relapsed/refractory ALL and LLy. Preliminary results from the ongoing Children's Oncology Group (COG) AINV18P1 trial are reported. Methods: Patients aged 1-30 years with first or greater isolated or combined marrow relapses of T-ALL or T-LLy and second or greater relapses of B-ALL or B-LLy are eligible for COG AINV18P1 (NCT03792256). Patients with refractory disease with at least 2 prior induction attempts or first relapse refractory to at least one prior re-induction attempt are also eligible. This is a 2-part study conducted at selected COG sites. In Part 1 of the study (dose determination), palbociclib was administered orally once daily for 21 consecutive days, first as a single agent (days 1-3) and subsequently in combination with 4-drug re-induction chemotherapy (Table 1). Study treatment consisted of a single cycle of therapy. A starting palbociclib dose of 50 mg/m2/dose was explored with one dose de-escalation (35 mg/m2/dose) using the rolling six design to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Part 2 (expansion cohort) is accruing 6 additional patients (pts) at the MTD for further assessment of the safety and feasibility of combination therapy. Dose-limiting toxicities (DLTs), defined as hematologic and non-hematologic toxicities exceeding baseline toxicities observed with re-induction chemotherapy alone, are assessed throughout the treatment cycle. The primary aims of the study are safety, determination of the MTD/RP2D and assessment of palbociclib pharmacokinetics (PK). Secondary aims are assessment of the biological and preliminary clinical activity of combination therapy in this patient population. Results: As of June 30, 2020, 8 pts have enrolled; 6 in Part 1 and 2 in Part 2. All 6 pts in Part 1 completed one full cycle (32 days) of protocol therapy. The median (range) age of pts is 10 (6-21) years and 62% are male (Table 2). Three pts had T-ALL, 4 B-ALL and 1 T-LLy. Pts received a median (range) of 2 (1-6) prior chemotherapy regimens and 2 pts underwent prior allogeneic stem cell transplant. All 6 pts in Part 1 experienced grade 3-4 toxicities, most commonly hematologic: anemia (3 pts), neutropenia (5 pts) and thrombocytopenia (4 pts). Three pts had grade 3 infections and one pt had grade 3 hyperbilirubinemia, which resolved. No hematologic or non-hematologic DLTs were observed at the 50 mg/m2/dose of palbociclib in Part 1 and accrual to the expansion phase of the trial at this dose continues. PK and pharmacodynamic studies assessing cell cycle inhibition are also ongoing. Conclusions: Palbociclib in combination with 4-drug re-induction chemotherapy is safe and well tolerated in children and young adults with relapsed/refractory ALL and LLy. Further assessment of the feasibility and activity of combination therapy is ongoing in the expansion phase of this trial. Disclosures Raetz: Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Teachey:La Roche: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sobi: Consultancy. Norris:Merck: Other: Travel funding. Gore:Amgen, Novartis, Roche: Membership on an entity's Board of Directors or advisory committees. Loh:Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Institutional Research Funding. OffLabel Disclosure: This study includes off-label use of palbociclib in children and young adults with relapsed/refractory acute lymphoblastic leukemia and lymphoblastic lymphoma.

Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8+ T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1+Tim-3+ CD8+ T cells were deficient in their ability to produce IFN-γ, TNF-α, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3+PD-1- KO CD8+ T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8+ T cells. Whereas blocking the PD-1/PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing—albeit not eliminating—both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8+ T-cell exhaustion in patients with hematologic malignancies such as advanced AML.

Tumor-induced immune defects can weaken host immune response and permit tumor cell growth. In a systemic model of murine acute myeloid leukemia (AML), tumor progression resulted in increased regulatory T cells (Treg) and elevation of program death-1 (PD-1) expression on CD8+ cytotoxic T cells (CTLs) at the tumor site. PD-1 knockout mice were more resistant to AML despite the presence of similar percentage of Tregs compared with wild type. In vitro, intact Treg suppression of CD8+ T-cell responses was dependent on PD-1 expression by T cells and Tregs and PD-L1 expression by antigen-presenting cells. In vivo, the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs. Anti–PD-L1 monoclonal antibody treatment increased the proliferation and function of CTLs at tumor sites, reduced AML tumor burden, and resulted in long-term survivors. Treg depletion followed by PD-1/PD-L1 blockade showed superior efficacy for eradication of established AML. These data demonstrated that interaction between PD-1 and PD-L1 can facilitate Treg-induced suppression of T-effector cells and dampen the antitumor immune response. PD-1/PD-L1 blockade coupled with Treg depletion represents an important new approach that can be readily translated into the clinic to improve the therapeutic efficacy of adoptive AML-reactive CTLs in advanced AML disease.

Tumor-induced immune suppression can permit tumor cells to escape host immune resistance. To elucidate host factors contributing to the poor response of adoptively transferred tumor-reactive cytotoxic T lymphocytes (CTLs), we used a systemic model of murine acute myeloid leukemia (AML). AML progression resulted in a progressive regulatory T-cell (Treg) accumulation in disease sites. The adoptive transfer of in vitro–generated, potently lytic anti–AML-reactive CTLs failed to reduce disease burden or extend survival. Compared with non–AML-bearing hosts, transferred CTLs had reduced proliferation in AML sites of metastases. Treg depletion by a brief course of interleukin-2 diphtheria toxin (IL-2DT) transiently reduced AML disease burden but did not permit long-term survival. In contrast, IL-2DT prevented anti-AML CTL hypoproliferation, increased the number of transferred CTLs at AML disease sites, reduced AML tumor burden, and resulted in long-term survivors that sustained an anti-AML memory response. These data demonstrated that Tregs present at AML disease sites suppress adoptively transferred CTL proliferation, limiting their in vivo expansion, and Treg depletion before CTL transfer can result in therapeutic efficacy in settings of substantial pre-existing tumor burden in which antitumor reactive CTL infusion alone has proven ineffective.

Abstract 4307 DNA hypermethylation and histone de-acetylation are mechanisms involved in gene expression silencing and subsequent drug resistance in leukemia. Agents which target these mechanisms have been investigated in myeloid malignancies but few studies have addressed whether they have a role in lymphoid leukemia. We conducted a clinical trial evaluating the tolerability and efficacy of Decitabine and Vorinostat followed by chemotherapy for relapsed/refractory acute lymphoblastic leukemia (ALL). Patients 0–60 years of age were eligible. Protocol therapy included Decitabine (15mg/m2 iv) and Vorinostat (230mg/m2 PO div BID) days 1–4 followed by vincristine, prednisone, PEG-Asparaginase and doxorubicin. Fourteen patients with a median age of 16 (range, 3 – 54) years have been enrolled to date. Five patients failed prior allogeneic transplantation. The most common non-hematological toxicities (grade >3) at least possibly related to Decitabine and/or Vorinostat were infection (grade 3; n=6) and fever (grade 3; n=3). There was a single grade 5 toxicity related to the chemotherapy backbone. Bone marrow samples (n=11) were evaluated using the DNA LINE global methylation analysis. A decrease in total methylation was found in 10 patients comparing baseline and day 5 (4/10 showing significant reductions in hypermethylation (T-test p Disclosures: Off Label Use: decitabine for relapsed ALL vorinostat for relapsed ALL.

Abstract 4126 Background: Childhood myelodysplastic syndrome (MDS) is a rare, heterogeneous disorder that is clinically distinct from adult MDS. Hematopoietic stem cell transplant (HSCT) is the treatment of choice, but there is no consensus regarding patient, disease, or treatment-related factors that predict outcomes after HSCT. Materials and Methods: We performed a retrospective review of 37 consecutive pediatric patients who received allogeneic HSCT for MDS at the University of Minnesota Amplatz Children's Hospital between 1990 and 2010. The median age at transplant was 11 years (range 1–21 years). Twenty patients had primary (de novo) MDS and 17 had secondary MDS (4 treatment-related, 8 with preceeding idiopathic aplastic anemia, 3 with Schwachman Diamond syndrome, and 2 familial). Those with Fanconi Anemia were excluded. Cytogenetics at diagnosis included monosomy 7 (n=21), trisomy 8 (n=7), normal/other (n=8). Thirty-one had refractory cytopenia (RC) and 6 had refractory anemia with excess blasts (RAEB) according to the modified WHO MDS classification. Patients were scored according to the International Prognostic Scoring System as low risk (n=1), intermediate-1 (Int-1; n=15), intermediate-2 (Int-2; n=21), or high risk MDS (n=0). Six patients received pre HSCT chemotherapy. Immediately prior to transplant, 27 had Results: Neutrophil engraftment occurred in 89% (95%CI 77–97%) of patients at a median of 23 days (range 12–40). Patients transplanted after year 1999 were more likely to engraft (RR 2.27; 95% CI 1.06–4.88, p=.04). Overall survival (OS) was 70% (95%CI 53–82%) and 53% (95% CI 36–68%) at 1 and 3 years. In multivariate analysis (MVA), OS at 1 year was increased in patients who did not receive pre HSCT chemotherapy (RR of death 0.04; 95% CI 0–0.50, p=.01) and decreased in those with an IPSS score of Int-2 (RR of death 11.96; 95%CI 1.29–110.74, p=.03). Disease free survival (DFS) was 62% (95%CI 44–75%) and 48% (95% CI 31–63%) at 1 and 3 years. In MVA, factors associated with improved DFS at 3 years include having secondary MDS (RR of death or relapse 0.13; 95% CI 0.02–0.69 p=.02), undergoing HSCT after 1999 (RR 0.06; 95% CI 0.01–0.70, p=.02), not receiving pre HSCT chemotherapy (RR 0.06, 95% CI 0.01–0.36, p Conclusions: Our results suggest that in order to achieve optimal outcomes, children with MDS should be referred for allogeneic HSCT soon after diagnosis and that unlike in adult MDS, pre HSCT chemotherapy does not appear to improve outcomes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3377 Poster Board III-265 Introduction: Allo-HCT with best available donor for children with Philadelphia positive (Ph+) ALL has previously been considered standard practice. Since the introduction of imatinib into the treatment of this disease, the role of allo-HCT is more uncertain. Patients and Methods: We investigated the impact of remission status, graft source, and imatinib use on transplant outcomes for thirty-seven children with Ph+ ALL who received an allo-HCT at the University of Minnesota between 1990 and 2006. The median age at HCT was 7.47 (range; 1.4 -16.4) years. Thirteen patients received imatinib therapy pre and/or post-HCT (imatinib group) and 24 patients, received either no imatinib (n=23) or only after post-HCT relapse (n=1) (non-imatinib group). Results: There was no difference in disease-free survival (DFS) or relapse between the imatinib and non-imatinib groups at 3 years (62% / 15% vs. 53% / 26%; p=0.99; 0.81, respectively). There was no significant difference in transplant outcomes between matched related donor or unrelated donor (umbilical cord blood or matched unrelated marrow) recipients whereas patients receiving allo-HCT in first remission (CR1) had superior DFS and less relapse compared to patients transplanted in ≥CR2 (71% / 16% vs. 29% / 36%; p=0.01; p=0.05). Conclusions: Based on this retrospective analysis at a single institution, the use of imatinib either pre and/or post-transplant does not appear to significantly impact outcomes for children with Ph+ ALL and allo-HCT with the best available donor should be encouraged in CR1. Disclosures: No relevant conflicts of interest to declare.

Leukemia involving rearrangements of the MLL gene is resistant to standard therapies and is often a fatal disease. MLL gene-rearrangements are commonly associated with infant-leukemia and secondary leukemias. New therapeutic strategies are needed to achieve longer survival and improve cure rates in patients with these and other refractory leukemias. Toll-like receptor (TLR) agonists are known as potent immune stimulatory agents that can elicit host anti-tumor effects in murine tumor models. We hypothesized that targeting TLRs expressed on leukemia cells with TLR agonists may have direct antitumor effects against leukemia cells. In this study, we investigated the effects of TLR agonists specific for TLR3, 4, 7, and 9, (i.e., polyinosine-polycytidylic acid (Poly(I:C)), monophosphoryl lipid A (MPL), imiquimod (IMQ), and CpG oligodeoxynucleotides (CpG ODN)), in MLL-AF9 knock-in mice that develop myeloid leukemia akin to human MLL-AF9 disease. In contrast to Poly(I:C), MPL, and CpG ODN, treatment of MLLAF9 cells with TLR7 agonist IMQ significantly increased the surface expression of CD40, CD54, CD80, and CD86 on MLL-AF9 cells in vitro. TLR7 mRNA and protein expression in MLL-AF9 cells were confirmed by real-time RT-PCR and intracellular staining/FACS analysis. Most importantly, TLR7 agonist strongly inhibited the in vitro MLL-AF9 cells in a drug dose- and treatment time-dependent manner. Whereas MLL-AF9 cells proliferated rapidly in culture with more than 40-fold increase of cell number in 5 days, the addition of IMQ at 5 mcg/ml fully inhibited the growth and induced profound apoptosis of MLL-AF9 cells with less than 2% of leukemia cells left at day 5 of culture. IMQ-mediated apoptotic death of MLL-AF9 was confirmed by viable cell counts, TMRE staining, Western blots and intracellular staining of the cleavage of caspases and PARP. Preincubation of MLLAF9 cells with caspases 8 and 10 inhibitors effectively blocked IMQ-induced apoptosis and sustained the proliferation of leukemia cells in cultures. To further determine the intracellular pathways engaged by IMQ, microarray gene expression profiles of 24-hour IMQ-treated vs. untreated MLL-AF9 cells were compared. Gene Set Enrichment Analysis (GSEA) showed that IMQ treatment resulted in up-regulated expression of a set of proapoptotic genes (e.g., p53, Bax, caspase 8, Apaf-1, etc) involved in apoptotic pathways. To determine whether IMQ pre-treatment of MLL-AF9 cells would prolong survival due to an apoptotic effect, cohorts of NOD-scid IL2Rgamma mice were i.p. injected with a lethal dose of MLL-AF9 cells with or without pre-incubation with IMQ. Mice receiving 5×106 untreated MLL-AF9 cells resulted in uniform lethality in 4 weeks. In contrast, mice receiving the same lethal dose of MLL-AF9 cells pretreated with IMQ had a significant prolonged survival, confirming in vitro findings that IMQ-treated MLL-AF9 cells undergo apoptosis. Administration of IMQ (daily i.p. injection at 1 mg/kg for 5 days) strongly inhibited leukemia cells growth and significantly prolonged the survival time of MLLAF9 mice. Flow cytometry results confirmed that residual MLL-AF9 cells recovered from IMQ treated mice were apoptotic and had up-regulated expression of cleaved caspases and PARP. In summary, our results demonstrate that TLR7 targeting of MLL-AF9 cells can directly induce apoptosis of MLL-AF9 cells in vitro and in vivo, providing new insights into the TLR-targeted therapy of refractory or relapsed leukemias.

Regulatory T-cells impede efficient immuno-surveillance and tumor clearance. Therapeutic Treg reduction with anti-CD25Ab can lead to improved survival in murine tumor models, but due to its long half-life, anti-CD25mAb may deplete anti-tumor reactive T-cells that are generated or expanded in Treg-depleted recipients. Tregs can also be targeted using an immunoconjugate consisting of IL-2 linked to diptheria toxin (IL- 2DT), that has a shorter half-life. Clinical trials with IL-2DT have had variable results in stimulating anti-tumor immune responses in the context of large tumor burdens. To determine whether responses with IL-2DT can be achieved in AML patients with minimal residual disease after induction chemotherapy, C57BL/6 (B6) mice were injected i.v. with moderately immunogenic luciferase- and DsRed-transduced murine AML cell line (C1498) that is MHC class I+II-. IL-2DT had no effects on in vitro tumor growth. After in vivo tumor injection, cohorts were given 1ug IL-2DT on days 0, 2 and 4 and monitored for tumor burden by Xenogen® luciferase imaging and survival. Untreated mice all died by d35. Whereas mice treated with IL-2DT vs. untreated controls initially showed no difference in tumor growth, tumor burden started to decrease by day 10 in treated mice. No tumor was observed in treated mice by d28, and all mice survived long term without relapse (p

17-AAG, an analogue of gelganamycin is an inhibitor of the molecular chaperone Hsp90, that results in apoptosis and inhibition of proliferation of myeloid or mixed lineage (MLL fusion gene induced) leukemia cells. The sensitivity to 17-AAG is highest in leukemia with FLT3 mutations, intermediate in wild type FLT3 and lowest in FLT3 negative leukemia cells. 17-AAG results in reduction of total levels of protein kinases FLT3, RAF, AKT and Chk1, a protein involved in DNA repair. Etoposide is a clinically effective agent in myeloid or mixed lineage leukemia therapy. Etoposide inhibits topoisomerase II and results in production of DNA double-strand breaks (DSBs). Normally these DSBs can be repaired by Rad51 via homologous recombination (HR). In DNA repair, Chk1 is required for HR through the interaction with Rad51. The effects of 17-AAG on Rad51 are unknown. Our current study evaluated the single and combined effects of 17-AAG and etoposide, and the mechanism of these effects in human leukemia cell lines with or without FLT3 mutations. Cell growth experiments using the MTT assay showed that the cell lines with MLL fusion genes and internal tandem duplication of FLT3 (ITD-FLT3) (Molm13 and MV4;11) were sensitive to both 17-AAG and etoposide. The IC50s for 17-AAG were 31 nM and 40 nM, respectively; the IC50s for etoposide were 37.7 nM and 45.6 nM, respectively. Wild-type FLT3 cells (HPB-Null and RS4;11) were less sensitive to both 17-AAG and etoposide; the IC50s for 17-AAG were 470 nM and 700 nM, respectively, and the IC50s for etoposide were 72.2 nM and 101.5 nM, respectively. The combination effects of 17-AAG and etoposide on cell proliferation were analyzed using Combination Index method (CalcuSyn software). Importantly, we observed synergistic inhibitory effects in FLT3-ITD cells (MV4;11 and Molm13) but only additive effects in wild type FLT3 cells (HPB-Null and RS4;11). Cell cycle analysis of MV4;11 and Molm13 cells showed that 17-AAG increased cells in G0/G1 phase after a 24 hrs treatment, while etoposide induced G2/M arrest only. Combined treatment with 17-AAG and etoposide results in Go/G1 arrest before the cells enter S phase, as with 17-AAG alone. Western-blotting showed that 17-AAG inhibited FLT3, Chk1 and in novel results Rad51 in ITD-FLT3 leukemia cells. To address the importance of FLT3 mutations on DNA repair proteins, Chk1 and Rad51, we used small interfering RNA (siRNA) targeted to FLT3. The results showed that Chk1 and Rad51 are dependent on constitutively activated FLT3 expression in ITD-FLT3 cells. In conclusion, the combination of 17-AAG with etoposide results in synergistic cellular inhibitory effects in ITD-FLT3 leukemia cells. The mechanism of the synergistic effects was found to be in part the result of inhibitory actions of 17-AAG on FLT3 dependent DNA repair proteins, Chk1 and in new findings Rad51 which are required for the repair of DNA damage induced by etoposide. 17-AAG, which is currently in clinical trials, combined with a topoisomerase II inhibitor, such as etoposide, has the potential to enhance therapeutic efficacy, particularly in high risk myeloid or mixed lineage leukemias with FLT3 mutation.