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PURPOSE The US Food and Drug Administration–expanded access program (EAP) uses a single patient use (SPU) mechanism to provide patient access to investigational agents in situations where no satisfactory or comparable therapy is available. Genomic profiling of de novo and relapsed or refractory childhood cancer has led to increased identification of new drug targets in the last decade. The aim of this study is to examine the SPU experience for genomically targeted therapies in patients with pediatric cancer. PATIENTS AND METHODS All genomically targeted therapeutic SPUs obtained over a 5-year period were evaluated at four large pediatric cancer programs. Data were collected on the type of neoplasm, agents requested, corresponding molecularly informed targets, and clinical outcomes. RESULTS A total of 45 SPUs in 44 patients were identified. Requests were predominantly made for CNS and solid tumors (84.4%) compared with hematologic malignancies (15.6%). Lack of an available clinical trial was the main reason for SPU initiation (64.4%). The median time from US Food and Drug Administration submission to approval was 3 days (range, 0-12 days) and from Institutional Review Board submission to approval was 5 days (range, 0-50 days). Objective tumor response was seen in 39.5% (15 of 38) of all evaluable SPUs. Disease progression was the primary reason for discontinuation of drug (66.7%) followed by toxicity (13.3%). CONCLUSION SPU requests remain an important mechanism for pediatric access to genomically targeted agents given the limited availability of targeted clinical trials for children with high-risk neoplasms. Furthermore, this subset of SPUs resulted in a substantial number of objective tumor responses. The development of a multi-institutional data registry of SPUs may enable systematic review of toxicity and clinical outcomes and provide evidence-based access to new drugs in rare pediatric cancers.

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation–dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Risk stratification and appropriate treatment selection for children with precursor B-acute lymphoblastic leukemia (B-ALL) have improved outcomes. We report the case of a 4-year-old male with a lymphomatous cavernous sinus mass, a previously undescribed presentation of newly diagnosed hyperdiploid B-ALL. Few case reports in the literature describe lymphomatous involvement in this region, but none are associated with pediatric B-ALL. This case presented unique treatment and risk assignment challenges given the intracranial location of this tumor and proximity to the central nervous system.

As patient-derived xenograft (PDX) models of acute myeloid leukemia (AML) become increasingly common tools for preclinical evaluation of targeted therapies it becomes important to consider the fidelity with which this system recapitulates the disease state found in patients. Gene expression profiling of patient blasts has been successfully used to identify distinct subtypes of AML to uncover sub-type specific vulnerabilities and to predict response to therapy and outcomes. Currently, there is little information regarding how well PDX models of AML mimic global gene expression patterns found in patients. In order to address this point, we performed detailed RNA-Seq analysis of data obtained from a diverse series of pediatric AML PDXs, separately and compared to primary patient data. When unsupervised clustering was applied to the PDX sample dataset, we found grouping associated with KMT2A (MLL) gene status. Additionally, in combined analysis, PDX samples were found to align with primary patient samples harboring similar genetics. We found a strong correlation of expression levels of nearly all expressed transcripts in PDX and patient datasets thus demonstrating faithful recapitulation of gene expression signatures. Furthermore, paired patient/PDX samples showed strong concordance, suggesting retention of sample-specific gene expression in immune deficient mice. Comparisons of PDX models propagated in NOD/SCID/IL2rg-/-(NSG) mice compared to NSG mice with transgenic expression of human SCF, GM-CSF, and IL-3 (NSGS) revealed minimal differences related to increased JAK/STAT and macrophage activation pathways in NSGS. Additionally, a unique RAM immunophenotype associated expression signature pointed to discovery of cryptic CBFA2T3-GLIS2 rearrangement as the mechanistic driver mutation in two PDX models. Based on the relatively high BCL2 mRNA in these models, we tested the efficacy of venetoclax in combination with CPX-351 which resulted in reduced leukemia burden and prolonged survival. These results validate the PDX system as surrogate of the molecular signatures in high-risk pediatric AML and highlight this system’s utility for pre-clinical therapeutic discovery, especially in very rare subtypes of disease.

The negative regulator of p53, MDM2, is frequently overexpressed in acute myeloid leukemia (AML) that retains wild-type TP53 alleles. Targeting of p53-MDM2 interaction to reactivate p53 function is therefore an attractive therapeutic approach for AML. Here we show that an orally active inhibitor of p53-MDM2 interaction, DS-5272, causes dramatic tumor regressions of MLL-AF9-driven AML in vivo with a tolerable toxicity. However, the antileukemia effect of DS-5272 is markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. In relation to this, DS-5272 triggers immune-inflammatory responses in MLL-AF9 cells including upregulation of Hif1α and PD-L1, and inhibition of the Hif1α-PD-L1 axis sensitizes AML cells to p53 activation. We also found that NK cells are important mediators of antileukemia immunity. Our study showed the potent activity of a p53-activating drug against AML, which is further augmented by antitumor immunity., MDM2 is frequently overexpressed in acute myeloid leukaemia leading to p53 inactivation. Here, the authors are demonstrating that an inhibitor of p53-MDM2 interaction, DS-5272, induce in vivo tumour regression through immune response regulation.

No guidelines exist for which intensive chemotherapy regimen is best in pediatric or young adult patients with high-risk posttransplant lymphoproliferative disorder (PTLD). We retrospectively reviewed patients with PTLD who received interval-compressed short-course etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin (SC-EPOCH) regimens at our institution. Eight patients were included with median age of 12 years. All patients achieved a complete response with a manageable toxicity profile. Two patients developed second, clonally unrelated, EBV-positive PTLD and one patient had recurrence at 6 months off therapy. No graft rejection occurred during therapy. All eight patients are alive with median follow-up of 29 months.

Immune therapies such as blinatumomab, CD19-directed bispecific CD3 T-cell Engager (BiTE), have resulted in significant improvements in outcomes for relapsed B-cell acute lymphoblastic leukemia (B-ALL). However, up to half of blinatumomab treated patients do not respond completely or relapse after therapy. As a result, there is a need to identify potential strategies to improve the efficacy of BiTE therapy. The anti-PD-1 antibody pembrolizumab has been shown to successfully activate T cells against a wide range of cancer types. Here, we tested the ability of umbilical cord blood (UCB) reconstituted mice to respond to blinatumomab therapy with or without concurrent pembrolizumab treatment. Humanized mice were engrafted with patient-derived xenograft (PDX) cells derived from pediatric and adolescent/young adult (AYA) B-ALL patients who had either failed to achieve remission with negative minimum residual disease (MRD negative) or experienced a relapse. Mock-treated humanized mice engrafted with PDX cells efficiently developed overt disease within 30 days of engraftment of B-ALL. However, single agent therapy with either blinatumomab or pembrolizumab reduced disease burden in engrafted mice, with some mice observed to be MRD negative after the 28-day treatment course. Combination therapy significantly improved the percentage of MRD negative mice and improved long-term survival and cure rates as compared to mice that were given blinatumomab alone. Importantly, no benefits were observed in treated mice that lacked human immune cell reconstitution. These results indicate that UCB-humanized NRGS mice develop activatable immune function, and UCB-humanized PDX leukemia models can be used in preclinical studies to evaluate specificity, efficacy, and cooperativity of immune therapies in B-ALL.

FOXM1, a known transcription factor, promotes cell proliferation in a variety of cancer cells. Here we show that Foxm1 is required for survival, quiescence and self-renewal of MLL-AF9 (MA9)-transformed leukemia stem cells (LSCs) in vivo. Mechanistically, Foxm1 upregulation activates the Wnt/β-catenin signaling pathways by directly binding to β-catenin and stabilizing β-catenin protein through inhibiting its degradation, thereby preserving LSC quiescence, and promoting LSC self-renewal in MLL-rearranged AML. More importantly, inhibition of FOXM1 markedly suppresses leukemogenic potential and induces apoptosis of primary LSCs from MLL-rearranged AML patients in vitro and in vivo in xenograft mice. Thus, our study shows a critical role and mechanisms of Foxm1 in MA9-LSCs, and indicates that FOXM1 is a potential therapeutic target for selectively eliminating LSCs in MLL-rearranged AML., FOXM1 is a known transcription factor which promotes cell proliferation in cancer cells. Here, the authors show that FOXM1 is required for the maintenance of quiescence and self-renewal of leukemia stem cells in MLL-AF9-rearranged acute myeloid leukemia patient and mouse models.

Aberrant activation of RHO guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in transformation and tumorigenesis. Consequently, a small molecule inhibitor of RhoGEF activities can be used as an anti-cancer drug. Herein, we used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that IODVA1 binds tightly to VAV3, inhibits RAC activation and signaling, and increases pro-apoptotic activity in BCR-ABL1-transformed cells only. Consistent with this mechanism of action, both VAV3-deficient leukemic cells and mouse models of BCR-ABL1 leukemia do not respond to IODVA1. IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal by decreasing RAC signaling in vivo. Importantly, IODVA1 is superior to standard of care dasatinib and ponatinib at prolonging the survival of PDX models of relapsed pediatric Ph+ and TKI-resistant Ph+ B-ALL with commonly found genetic mutations especially after treatment withdrawal. Cells representing pediatric ALL patients with diverse genetic lesions are highly sensitive to IODVA1 ex vivo and this sensitivity is VAV3-dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapeutic agent. Citation Format: Nicolas N. Nassar, Shailaja Hegde, Kark Wunderlich, Yuan Lin, Reza Ahmadian, William Seibel, Yi Zheng, Benjamin E. Mizukawa, Lisa Privette Vinnedge, Jose A. Cancelas. Inhibition of the RAC Activator VAV3 by the small molecule IODVA1 impedes RAC signaling & overcomes resistance to tyrosine kinase inhibition in lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB198.

Summary Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3–91·5] and overall survival was 95·4% (95% CI 91·8–99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1–3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P

We have previously characterized an acute myeloid leukemia (AML) chemotherapy model for SCID-based immune deficient mice (NSG and NSGS), consisting of 5 days of cytarabine (AraC) and 3 days of anthracycline (doxorubicin), to simulate the standard 7+3 chemotherapy regimen many AML patients receive. While this model remains tractable, there are several limitations, presumably due to the constitutional Pkrdcscid (SCID, severe combined immune deficiency) mutation which affects DNA repair in all tissues of the mouse. These include the inability to combine preconditioning with subsequent chemotherapy, the inability to repeat chemotherapy cycles, and the increased sensitivity of the host hematopoietic cells to genotoxic stress. Here we attempt to address these drawbacks through the use of alternative strains with RAG-based immune deficiency (NRG and NRGS). We find that RAG-based mice tolerate a busulfan preconditioning regimen in combination with either AML or 4-drug acute lymphoid leukemia (ALL) chemotherapy, expanding the number of samples that can be studied. RAG-based mice also tolerate multiple cycles of therapy, thereby allowing for more aggressive, realistic modeling. Furthermore, standard AML therapy in RAG mice was 3.8-fold more specific for AML cells, relative to SCID mice, demonstrating an improved therapeutic window for genotoxic agents. We conclude that RAG-based mice should be the new standard for preclinical evaluation of therapeutic strategies involving genotoxic agents.

TPS7052 Background: Despite significant advances in therapy for acute myeloid leukemia (AML), 30-40% of young patients will relapse, after which prognosis is poor. In young patients, curative-intent salvage therapy involves intensive re-induction followed by hematopoietic stem cell transplant. Recently, the COG Phase II study of CPX-351 (liposomal cytarabine:daunorubicin, Vyxeos™) in pediatric patients with AML in first relapse (NCT02642965) demonstrated a CR/CRi rate of 81.3%. Separately, our first-in-pediatrics CPX-351 Phase I (NCT01943682) showed 48% in a heavily pre-treated pediatric cohort with multiply relapsed and refractory (R/R) AML. Our integrated pilot study of single cell RNA sequencing (scRNA-seq) done before, during, and after CPX-351 showed p53 targets over time with enrichment for genes regulating apoptosis (ex.: FAS, BAX), suggesting blasts may be primed for apoptosis following CPX-351. Venetoclax is a small molecule inhibitor of the anti-apoptotic protein BCL-2, a regulator of apoptotic balance in some leukemias. Based on our preclinical data, we developed a Phase I study to investigate venetoclax with CPX-351 for the treatment of young patients with R/R acute leukemias. Methods: The V2 Trial (NCT03826992) is a single-institution Phase I study to evaluate the safety and tolerability of venetoclax with CPX-351 in patients ages 1-39 years with R/R acute leukemias. Inclusion diagnoses include AML, mixed phenotype acute leukemia (MPAL), KMT2A-rearranged acute lymphoblastic leukemia (ALL), and T-ALL. Exclusion criteria include CNS status 3, bone marrow failure syndromes, and prior cardiotoxic exposures above acceptable risk thresholds. Subjects receive a single course of CPX-351 at the FDA approved adult dose on Days 1, 3, 5 with concurrent daily venetoclax. In the dose exploration phase, venetoclax dosing is 400 mg daily (or allometrically-scaled equivalent) for 21 (Dose Level 0) or 14 days (Dose Level -1) using a rolling 6 design. Primary endpoints are determination of the recommended phase 2 dose of venetoclax in combination with CPX-351 and description of toxicities. Secondary endpoints include estimations of CR/CRp/CRi +/- MRD negativity in the context of a phase I study and evaluation of therapy-related cardiac dysfunction. Correlative studies include analysis of venetoclax pharmacokinetics with concomitant CPX-351. At the initial dose level, DLT were encountered and the study is now continuing enrollment at Dose Level -1. Clinical trial information: NCT03826992.

Introduction: The Food and Drug Administration (FDA) expanded access program uses a single-patient use (SPU) mechanism to provide patient access to investigational agents prior to FDA approval and outside clinical trials, in situations where no satisfactory or comparable therapy is available. Genomic profiling of both newly diagnosed and relapsed/refractory childhood cancer has increased in the last decade, resulting in identification of new drug targets for pediatric malignancies. Recently, a review of SPU use in adult and pediatric patients at a single institution (Feit, JAMA Oncology 2019) showed that a markedly higher percentage of pediatric patients receive access through SPUs compared with adult patients, identifying this as an important means of pediatric drug access. However, little is known about the pediatric use and efficacy of SPUs in children. The aim of this study is to examine the SPU experience in pediatric and adolescent and young adult (AYA) cancer patients—specifically obtained for targeted therapies at three large pediatric cancer centers. Methods: All therapeutic SPUs obtained between January 1, 2014 and January 1, 2019 were evaluated at the Aflac Cancer & Blood Disorders Center (Atlanta, GA), Cincinnati Children’s Hospital (Cincinnati, OH), and the Memorial Sloan Kettering Cancer Center (New York, NY). Data were collected on the type of malignancy, agents requested, and corresponding molecularly informed targets, if applicable. Results: A total of 61 SPUs were approved in the five-year period, with 34 (55%) of them specific for access to agents based on somatic tumor mutations identified by genomic profiling. Among the disease groups, SPUs were most often requested for tumors affecting the central nervous system (CNS) (48%), followed by solid tumors (bone, liver, and kidney tumors) (26%), hematologic malignancies (leukemia/lymphoma) (21%), and other rare tumors (5%). Kinase inhibitors were the most frequently requested agents in the genomically defined category (n=34), specifically, FGFR (fibroblast growth factor receptor) inhibitors followed by drugs targeting NTRK 1/2/3 (tropomyosin receptor kinase (Trk) receptors). Most patients within this genomically targeted group (18/34) are currently receiving therapy with these agents. Conclusions: We found that SPUs represent an important means of access to therapeutic agents in the pediatric and AYA populations, with more than half of all SPUs based on rearrangements identified by genomic profiling. A broad range of agents were requested across CNS, solid tumor, and hematologic types. Furthermore, more than half of the patients remain on their respective SPU-approved drug. We are currently performing a more detailed analysis of clinical responses, as well as a time analysis from SPU initiation to approval for all patients in this cohort. Establishment of this cohort of patients across institutions will serve as the basis for a formal registry of pediatric SPUs, which will enable us to study their use and efficacy over time. Citation Format: Himalee Sabnis, Benjamin Mizukawa, Julia Glade-Bender, Jason Fangusaro, Stephen Roberts, Chanta Whitlow, Marilyn Winchester, Maureen O’Brien, Laura Agresta, Brian Turpin, Daniel Wechsler, Sharon Castellino, Neerav Shukla. Targeted therapies for children and young adults with cancer: Single-patient use (SPU) experience at three large pediatric cancer programs [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A50.

e22502 Background: Hypomethylating agents (HMAs) have favorable toxicity profiles and anti-leukemia activity in adult AML/MDS. We previously reported similar activity in eight pediatric patients (Phillips et al., British Journal of Hematology 2013), however, it remains unclear which factors influence outcome and sustainability of response. We sought to update our clinical experience and determine molecular correlates of HMA response and relapse with the goal of identifying potential biomarkers and physiologic insights. Methods: Clinical data was retrospectively collected through chart review from 2012 to 2017. Cryopreserved patient bone marrow was obtained from our institution’s biorepository under an IRB-approved protocol. Leukemic blasts were enriched by FACS, and genomic DNA was extracted and subjected to reduced representation bisulfite sequencing (RRBS). Differentially methylated regions (DMRs) were calculated using MethylKit. Results: Fourteen patients were treated with hypomethylating agents. Median age was 9.5 years (range 2-34). Two patients (14%) had germline TP53 mutations, secondary MDS/AML, and achieved complete response (CR). Four patients (29%) had monosomy 7, with 2 CR (50%) and 2 partial responses (PR). Contrarily, in the non-monosomy 7 patients, only 1 patient achieved a CR, 2 PR, and one had stable disease. DMRs were identified by comparing diagnostic and relapse samples, as well as responsive and refractory specimens. As expected, there was global DNA hypomethylation following treatment with HMAs. Pathway analysis of DMRs identified in relapse vs diagnostic samples revealed promoter hypomethylation in processes related to DNA binding, transcription factor activity, and RNA biosynthesis, as well as promoter hypomethylation of polycomb repressive complex 2 targets and gene sets classically associated with histone H3 K27 trimethylation. Gene set enrichment analysis (GSEA) of relapse versus remission marrow samples demonstrated reduced methylation of promoter regions regulating DNA repair, responses to DNA damage, and adaptive immune signaling. Conclusions: Our data suggests that HMA response and relapses after treatment may exhibit unique biological signatures reflected in methylation changes. These findings can inform further hypothesis generation and potentially combination therapies.

Cdc42 was originally discovered as a key regulator of bud site assembly and polarity in S. cerevisiae. Recent genetic studies have shown that the function of Cdc42 in regulating cell polarity appears highly conserved from budding yeast to humans. The role of Cdc42 in hematopoietic cell transformation and leukemia progression has been studied in an acute myeloid leukemia model using the MLL-AF9 oncogene-induced transformation and a Cdc42 conditional gene-targeted mouse model. Here we describe the leukemia cell polarity and division symmetry assays in the context of leukemia cell fate determination.

Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia-AF9 (MLL-AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation. Stem Cells 2015;33:3635–3642

Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival. Nonetheless, the physiological function of RhoA in thymocyte development is not clear. By characterizing a conditional gene targeting mouse model bearing T cell deletion of RhoA, we show that RhoA critically regulates thymocyte development by coordinating multiple developmental events. RhoA gene disruption caused a strong developmental block at the pre-TCR checkpoint and during positive selection. Ablation of RhoA led to reduced DNA synthesis in CD4−CD8−, CD4+CD8−, and CD4−CD8+ thymocytes but not in CD4+CD8+ thymocytes. Instead, RhoA-deficient CD4+CD8+ thymocytes showed an impaired mitosis. Furthermore, we found that abrogation of RhoA led to an increased apoptosis in all thymocyte subpopulations. Importantly, we show that the increased apoptosis was resulted from reduced pre-TCR expression and increased production of reactive oxygen species (ROS), which may be because of an enhanced mitochondrial function, as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, and mitochondrial biogenesis in RhoA-deficient thymocytes. Restoration of pre-TCR expression or treatment of RhoA-deficient mice with a ROS scavenger N-acetylcysteine partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate, to our knowledge, for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis.

As a central regulator of cell polarity, the activity of CDC42 GTPase is tightly controlled in maintaining normal hematopoietic stem and progenitor cell (HSC/P) functions. We found that transformation of HSC/P to acute myeloid leukemia (AML) is associated with increased CDC42 expression and activity in leukemia cells. In a mouse model of AML, the loss of Cdc42 abrogates MLL-AF9-induced AML development. Furthermore, genetic ablation of CDC42 in both murine and human MLL-AF9 (MA9) cells decreased survival and induced differentiation of the clonogenic leukemia-initiating cells. We show that MLL-AF9 leukemia cells maintain cell polarity in the context of elevated Cdc42-guanosine triphosphate activity, similar to nonmalignant, young HSC/Ps. The loss of Cdc42 resulted in a shift to depolarized AML cells that is associated with a decrease in the frequency of symmetric and asymmetric cell divisions producing daughter cells capable of self-renewal. Importantly, we demonstrate that inducible CDC42 suppression in primary human AML cells blocks leukemia progression in a xenograft model. Thus, CDC42 loss suppresses AML cell polarity and division asymmetry, and CDC42 constitutes a useful target to alter leukemia-initiating cell fate for differentiation therapy.

Despite significant advances in AML therapy over the past decades, 30-40% of all young patients with AML will suffer a relapse, after which achieving long-term disease-free survival remains challenging. In pediatric and young adult patients with AML, curative-intent treatment at relapse involves intensive induction therapy to achieve CR followed by consolidation with HSCT. Historically, initial response rates to salvage therapy were limited. Recently, Children's Oncology Group presented results from a Phase II study of CPX-351 (liposomal cytarabine:daunorubicin at a synergistic ratio) in pediatric patients with AML in first relapse demonstrating an impressive CR/CRp/CRi rate of 81.3%. Separately, we presented data from our first-in-pediatrics CPX-351 Phase I trial showing 48% M1 marrow response in a population consisting of children with multiply relapsed and refractory (R/R) AML patients. We also obtained single cell RNA sequencing on study patients' peripheral blasts before, during, and after CPX-351 treatment (i.e.: Days 0, 1 and 5). Preliminary data showed that CPX-351 differentially impacts p53 targets within leukemic blast clusters over time, with an enrichment for genes regulating apoptosis (e.g.: FAS, BAX) in most clusters. This suggests that these blasts may be primed for apoptosis following CPX-351 treatment. Anti-apoptosis signaling in leukemias is an established mechanism of chemoresistance. Venetoclax is an orally available, small molecule inhibitor of anti-apoptotic protein BCL-2, which is frequently overexpressed in leukemias. Based on published preclinical data and single cell RNA sequencing data from our CPX-351 trial, we developed a Phase I study to investigate the combination of CPX-351 and venetoclax in the treatment of young patients with R/R acute leukemias. The V2 trial is a single-institution Phase I pilot study (NCT03826992) designed to test the safety and tolerability of combining venetoclax with CPX-351 in patients ages 1-39 years for the treatment of R/R acute leukemias. Inclusion diagnoses are based on preclinical sensitivities and include AML, MPAL, or AUL in first or greater relapse, or ETP-ALL, MLL-rearranged ALL, or T-ALL in second or greater relapse. Patients must have measurable disease. Exclusion criteria include CNS status 3, bone marrow failure syndromes, and prior anthracycline and mediastinal radiation exposures above acceptable cardiotoxicity risk thresholds. Subjects receive a single course of study therapy consisting of daily venetoclax and CPX-351 at the FDA approved dose for adults on Days 1, 3, and 5 (Fig. 1) with the goal of CRMRD- to allow for off-study HSCT. The venetoclax dose exploration phase begins with a starting dose of 400 mg (or the allometrically-scaled equivalent) for 21 days with a 3-day ramp to target dose. The dose exploration phase proceeds on a rolling 6 design and will require 4-12 patients to determine RP2D. Provided a tolerable RP2D is found, a dose expansion phase will open. Primary endpoints are determination of RP2D and description of the safety profile including time to hematologic recovery. Secondary endpoints are estimations of CR/CRp/CRi +/- MRD- within the context of a phase I study, and evaluation of cancer therapy-related cardiac dysfunction (CTRCD) rates. Correlative studies include evaluation of venetoclax PK with concomitant CPX-351 using modeling & simulation with a combined limited sampling and pediatric opportunistic PK sampling (POPS) strategy, and exploration of the correlations between BCL-2 family expression and pediatric tumor genetics with treatment response. Figure 1 Disclosures O'Brien: BTG: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; AbbVie: Research Funding; Amgen: Research Funding; BMS: Research Funding. Phillips:Novartis: Membership on an entity's Board of Directors or advisory committees. Vinks:Myriad Genetics: Consultancy, Patents & Royalties. Perentesis:Kurome Therapeutics: Consultancy. Absalon:Jazz Pharmaceuticals: Other: CPX-351 for clinical trial . OffLabel Disclosure: This clinical trial involves the off-label use of CPX-351 and venetoclax for relapsed/refractory pediatric and young adult leukemias.

Aberrant activation of Rho guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in cancer. Thus, small molecule inhibitor of RhoGEF activities can be used as a drug lead to treat leukemia and other malignancies. We have identified an active small molecule, IODVA1, in several xenograft mouse models of cancer including Ras-driven cancers. Here, we used cellular and mouse models of Ph+(BCR-ABL1) B-ALL to identify Vav3, the multi-domain tyrosine phosphorylation-dependent RacGEF as the target of the small molecule IODVA1. IODVA1 specifically reduces the proliferation and survival of p190-BCR-ABL but not of empty vector expressing human CD34+ blood cells. IODVA1 binds tightly to recombinant Vav3 (Kd= 400 nM) but not to Rac1 or to the RhoGEF LARG. In Ba/F3 cells expressing p190-BCR-ABL, IODVA1 inhibits Rac activation and signaling within minutes of exposure. The decrease in Rac activity is not due to the activation of p50GAP or RhoGDI1 and is accompanied by a decrease in the activity of the pro-survival effectors PAK, JNK, and 4EBP and an increase in pro-apoptotic BAD activity. IODVA1prevents leukemia-related death and eliminates the leukemia burden in a BCR-ABL-induced murine model with no apparent toxicity. It eradicates leukemic propagating activity assessed by serial transplantation. Most importantly, IODVA1 increases the survival of a mouse model of TKI-resistant p210-BCR-ABL1(T315I) B-ALL better than ABL1-TKI imatinib and eliminates leukemic burden. p210-T315 leukemic mice survive 45 days after treatment has ended. Vav3- and Rac1/Rac2-deficient leukemic cells do not respond to IODVA1 in colony formation assay consistent with Vav3 being IODVA1's target. A mouse model of BCR-ABL leukemia deficient in Vav3 also does not respond to IODVA1. Cells from PDX models representing pediatric ALL patients including Ph+ (BCR-ABL(T315I) were found to be highly sensitive to IODVA1 ex vivo. Based on the described mechanism of action, we propose that IODVA1 is an allosteric inhibitor of Vav3 that holds promise as an anti-leukemic agent. Disclosures Cancelas: Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Velico: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding.

Background: Resistance to chemotherapy-induced apoptotic death is a major mechanism responsible for the failure of AML therapies. Levels of anti-apoptotic proteins BCL2 and MCL1 are increased in relapsed AML samples. Venetoclax, a BH3 mimetic that binds to BCL-2, was recently granted accelerated approval for use in AML in combination with cytotoxic chemotherapy. However, MCL-1 and BCL-XL are known pathways of resistance to BCL-2 directed therapy. AMG-176 is a first-in-class MCL-1 specific inhibitor that induces specific and significant apoptosis-mediated toxicity against leukemia cell lines, tumor xenograft models, and primary patient samples. We compared anti-leukemic synergy of Venetoclax and AMG-176 together and individually with cytotoxic chemotherapy used in AML. We sought to identify optimal combinations for efficacy against varying molecular signatures of AML. Methods: To examine the potential of pairing Venetoclax with AMG-176 we performed a series of in vitro 8x8 dose response matrices on a panel of 11 leukemias spanning AML and Acute Lymphocytic Leukemia (ALL) (Table). This included 3 relapsed pediatric Patient Derived Xenograft (PDX) AML lines propagated in the presence of cytokines to better model inflammation, stem cell function, and niche derived support of leukemic blasts. An additional 8 established cell lines representing key AML & ALL molecular classes were employed. We used the Delta Bliss (DB) method for scoring matrices and report the DBsum, the summation of all of the synergistic(-) and antagonistic(+) well scores in an 8x8 matrix. Drugs tested in combinations with Venetoclax and/or AMG-176 included cytarabine and daunorubicin. Results: The Figure shows average DBSum for the Venetoclax+AMG-176 combination was -3.41 in the 8 commercial lines, whereas combining the anthracycline Daunorubicin+AMG-176 combination yielded a DBsum score of -0.34, with the score being skewed by a marked antagonism observed with the t(8;21) AML line Kasumi-1 with a DBSum score of 2.13. The 3 relapsed pediatric PDX derived AML lines continued the trend of strong synergy with the Venetoclax+AMG-176 combination with an average score of -3.68 with no observed antagonistic interactions. For context, a synergistic DBSum score of -2.33 would be considered noteworthy from a combination dataset of over >4000 discreet combinations. The Venetoclax+AMG-176 combination exhibited the highest degree of synergy broadly across AML and ALL molecular subgroups. Different patterns of cytotoxicity of combination of chemotherapy with BCL2 or MCL1 inhibition were observed in leukemias with varying molecular backgrounds and histology, including patterns of strong synergy, additivity or antagonism. However, leukemias carrying KMT2A rearrangements exhibited strong synergy or additivity of BCL2 or MCL1 combinations with either daunorubicin or cytarabine, regardless of histology. Conclusions: We identified broad synergy in high risk genetic subtypes of AML and ALL with the combination of BCL-2 and MCL-1 inhibition, and the effect was superior to either agent combined with chemotherapy individually. We identified potential genetic signatures associated with response to chemotherapy cytotoxicity with either Venetoclax or AMG-176. In vivo experiments are in progress to evaluate the efficacy of this combination and better determine genetic subtypes who would most benefit from this treatment approach. Figure Disclosures Perentesis: Kurome Therapeutics: Consultancy.

Therapies for pediatric acute myeloid leukemia (AML) remain unsatisfactory and generally do not incorporate molecularly-targeted agents aside from FLT3 inhibitors outside of the relapse setting. Patient-derived xenograft (PDX) models of AML are increasingly accessible for the preclinical evaluation of targeted therapies, though the degree to which these systems recapitulate the disease state as found in patients has not been well defined for AML. Gene expression profiling of patient blasts has been successfully used to discriminate distinct subtypes of AML, to uncover sub-type specific vulnerabilities, and to predict response to therapy and outcomes. We sought to systematically examine PDX models of pediatric AML for their ability to replicate global gene expression patterns and preserve mutational signatures found in patients. In addition, we conducted in-depth bioinformatic analyses of samples with cryptic CBA2T3-GLIS2 fusion generated by the inv(16)(p13.3q24.3) for identification of potential novel targeted therapies. We performed detailed analyses of RNA sequencing data from a diverse series of 24 pediatric AML PDX models established from samples obtained from patients with relapse and refractory disease. Initially we compared our PDX data against 49 selected relapse and refractory patient sample data files found in the NCI TARGET dataset of pediatric AML. When applying unsupervised hierarchical clustering to the PDX samples, we found that clustering was associated with MLL status. Clustering of the combined sets of samples by MLL status showed integration of samples according to mutation profile, regardless of data source (PDX or patient). The expression levels of all detectable transcripts were highly conserved between PDX and patient MLL-r samples. Separate analysis of each dataset yielded MLL specific gene lists that included a subset of overlapping genes which may point to a unique relapse and refractory pediatric MLL-r signature. This list contains several interesting new targets for further study. A subset of 12 PDX models were compared directly to the matched patient sample from which they were established. This analysis revealed strong similarity, with each PDX most closely related to its matched patient sample, suggesting retention of sample-specific gene expression in immune deficient mice. We set up our PDX models in NSG mice with transgenic expression of human myelo-supportive cytokines SCF, GM-CSF, and IL-3 in order to promote the most efficient and robust engraftment of precious patient material. In order to detect any skewing effects due to the host mouse strain, we compared NSGS PDX RNA sequencing data to 10 matched NSG PDX models. This comparison revealed consistent differences in only 9 transcripts, which were almost entirely related to increased JAK/STAT signaling and macrophage activation pathways in NSGS mice relative to NSG mice. Interestingly, during this analysis we observed a distinct PCA-driven clustering of a pair of PDX samples with previously clinically unidentified driver mutations. Reanalysis of the RNA sequencing data revealed evidence of a cryptic GLIS2 rearrangement (found in ~1% of pediatric AML cases) as the driver mutation, which was subsequently confirmed by RT-PCR in both samples. The unique CBFA2T3/GLIS2 RNA signature was mined to guide the composition of a focused 75-molecule in vitro drug screen against ex vivo PDX samples with an emphasis on the SHH, WNT, and BCL2 pathways. This screen identified the Wnt-C59 PORCN inhibitor as having specific activity against CBFA2T3/GLIS2+ AMLs. Further testing of C-59 in combinatorial studies revealed enhanced effects with the addition of the BCL2 inhibitor, venetoclax. In vivo experiments are currently underway to determine the pre-clinical efficacy of this novel combination. In summary, we found highly significant fidelity of gene expression in PDX models of relapse and refractory pediatric AML. Analysis of this dataset has led to several insights, including potential targeted therapies, highlighting how this system could be a valuable tool for discovery of novel targeted therapies, especially for very rare, distinct subtypes of disease. Disclosures Perentesis: Kurome Therapeutics: Consultancy.

Hematopoietic stem cells (HSC) mainly engage glycolysis while leukemia stem cells (LSC), such as in acute myeloid leukemia (AML), heavily rely on mitochondrial (Mt) respiration (i.e. oxidative phosphorylation, OxPhos) to fuel energy. Growing evidence suggests this metabolic reprogramming confers therapeutic vulnerabilities in AML. BCL2 is overexpressed in LSC from AML patients, while BCL2 inhibitors, such as venetoclax (VEN), have been shown to suppress OxPhos in LSC and to eradicate LSC. In clinical practice, half of the patients fail to respond to VEN. VEN is a costly medication and patients who are resistant to VEN forgo alternative treatment at that time. Prediction of the response to VEN and strategies to circumvent resistance are urgently needed. BCL2 is shown to increase Mt Ca2+ levels ([Ca2+]m), which enhance OxPhos through activation of the Ca2+-sensitive dehydrogenases within the tricarboxylic acid (TCA) cycle. G protein-coupled receptor 68 (GPR68) is a proton sensor, activating phospholipase C that leads to releasing of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and elevation of cytosolic Ca2+ levels ([Ca2+]c). This prompted us to examine the cooperative effect of GPR68 and BCL2 on the Ca2+/OxPhos pathway in AML, and particularly in LSC. Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells. We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance. In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN. Disclosures No relevant conflicts of interest to declare.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1− cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL.

Background: CPX-351 (Vyxeos®) is a liposomal nanoparticle of cytarabine and daunorubicin in a 5:1 molar ratio currently FDA approved for treatment of adults with newly-diagnosed therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes. The agent also has potent activity against acute lymphoblastic leukemia (ALL) in murine xenograft models [H. Carol et al. Pediatr Blood Cancer 2015;62: 65-71]. We conducted the first phase I/pilot study to evaluate the safety and describe the efficacy of CPX-351 in children and adolescents with relapsed or refractory (R/R) acute leukemia (NCT01943682). Methods: In the Dose Exploration Phase participants age 12 months to 21 years were eligible to receive a single course of CPX-351 administered once daily on Days 1, 3, and 5. Enrollment was initiated at the recommended dose for study in adults (Dose Level 1: 100 Units/m2/dose= cytarabine 100 mg/m2 and daunorubicin 44 mg/m2 per dose) with provisions to explore either a single dose escalation to 134 Units/m2/dose (Dose Level 2) or de-escalation to 67 Units/m2/dose. Response was determined after Day 28. The upper age for eligibility was increased to 30 years and an additional 18 subjects were enrolled into the Expanded Phase of the study. Results: Nine children and adolescents with R/R AML (n=6) or ALL (n=3) were enrolled and evaluable for toxicity in the Dose Exploration Phase. All 9 experienced Grade 3 or 4 hematologic toxicity including 8 with fever with neutropenia or infection. Dose Limiting Toxicity (DLT) was not observed in the 4 patients enrolled at Level 1 but Grade 3 pain/headache lasting greater than 48 hours did occur in 2 of the 5 patients treated at Dose Level 2. One patient with AML was unevaluable for response in this phase due to removal from protocol therapy to receive bone marrow transplantation while in aplasia. All others were evaluable for response. One patient with ALL had stable disease, 2 with AML achieved a complete remission (CR), and 1 with AML achieved a CR in blood and marrow but developed low level disease in the cerebral spinal fluid (CSF). One patient with mixed phenotype acute leukemia and 17 with R/R AML were subsequently enrolled into the Expanded Phase of the study to receive CPX-351 at Dose Level 1. CPX-351 treatment was well tolerated in this population with a similar toxicity profile observed in the Dose Exploration Phase. Dose limiting prolonged aplasia occurred in one patient but no other DLTs were observed. The response has yet to be determined in one patient. To date, seven patients with AML (41%) achieved a CR or complete remission with incomplete blood recovery (CRi). Another patient achieved a CR in marrow but with low level disease detected in the CSF similar to the patient described in the Dose Exploration phase. The remaining 9 patients failed to achieve a clinical response. Pharmacokinetic (PK) parameters of total cytarabine and daunorubicin were analyzed for 21 of these pediatric patients and the results were similar to those observed in adults. However, due to the small sample size of each age group, these interim PK results will need to be validated as data in pediatric patients become available from future clinical studies. Conclusions: CPX-351 100 Units/m2/dose on Days 1, 3, and 5 was well tolerated in this pediatric population and has promising activity in children and young adults with R/R AML. Additional CNS directed therapy is recommended for pediatric patients at high risk for failure in the CNS. Disclosures Phillips: Novartis AG: Consultancy. An:Jazz Pharmaceuticals: Employment.

Each case of Acute Myeloid Leukemia (AML) represents a unique ecosystem. Despite recent advances in our understanding of the genetic landscape of AML, this information remains insufficient to accurately match patients with targeted therapies. While pediatric and adult AML share phenotypic similarities, pediatric AML represents a genetically distinct disease from adult AML, and will benefit from independent genomic studies and novel therapeutic strategies. Real-time ex-vivo functional screening can identify mechanisms underpinning drug response and diversity between tumors, aiding in patient stratification. We established an in vitro drug screening system that incorporates cytokine signaling to better model inflammation, stem cell function, and niche derived support of leukemic blasts. This approach provided insight into variability between patients who currently would be placed on the same therapeutic regimen. Samples were acquired from 12 pediatric AML patients, after informed consent was obtained. Samples were enriched for blasts, and cultured in the presence of SCF, TPO, FLT3-L, IL-3, and IL-6 (KTF36). A panel of 38 drugs was selected from a larger screen of 1839 compounds done on commercially available hematological malignancy cell lines. Drugs included standard chemotherapy agents used in AML and drugs currently under clinical development. Cells were exposed to drugs for 72hrs. An MTS assay was performed and results reported as % of viable cells remaining, after normalization to vehicle control wells. Targeted DNA NGS sequencing of 406 genes, 31 introns, and RNA sequencing of 265 genes was performed for genetic characterization. In vitro drug screening revealed variations in drug sensitivity between samples and revealed time ex-vivo and cytokine milieu to be important factors affecting response of the same cells to the same drugs. Engraftment of long term cultures into immunodeficient mice produced aggressive disease in all cases, indicating robust support of stem cell function via addition of KTF36 cytokines. When possible, clinical response to therapy was compared with in vitro response to the same drugs. This screening approach highlighted well established agents that showed significant activity in highly refractory disease providing rationale for further clinical trial development. An unsupervised clustering showed drug sensitivity primarily correlated with the presence of MLL-X fusion, NRAS/KRAS and PTPN11 alterations. Linear Regression with interaction effects showed drug sensitivity/resistance to be highly selective for single/signature of specific molecular alterations. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by dysregulated immune activation. Primary HLH involves hereditary deficits in cytotoxic lymphocytes while secondary HLH is triggered by extrinsic factors. The HLH-2004 criteria are widely used for clinical diagnosis, yet their specificity for HLH or their ability to differentiate primary from secondary disease is unclear, potentially leading to inappropriate treatment. We describe several cases where fulfillment of HLH-2004 criteria obscured the diagnoses of underlying malignancies and delayed curative management. These issues are remedied without waiting for genetic testing results through an alternative diagnostic approach using flow cytometry-based immunologic assays and a thorough investigation for malignancy.

e19011Background: Each case of acute myeloid leukemia (AML) represents a unique ecosystem. Despite recent advances in our understanding of the genetic landscape of AML, this information remains ins...

11514 Background: Contemporary chemotherapy-based regimens provide cures for most pediatric & AYA cancers. However, for patients with relapsed/refractory malignancies, outcomes are poor & imply a distinct and aggressive biology. Identifying common themes in the molecular architecture & oncogenic mechanisms in these patients is a critical priority for drug development. We hypothesized that the molecular signature of cancers in these patients would be independent of histology. We also assessed the response to molecular alteration (MA)-targeted therapies. Methods: IRB-approved analysis of MAs in 306 relapsed/refractory pediatric & AYA malignancies (116 hematologic malignancies, 68 sarcomas, 46 neuroblastomas, 36 CNS, 14 liver, 9 renal, 17 other) was performed. DNA was analyzed for MAs (Foundation Medicine, Cambridge, MA; Univ of Washington, Seattle, WA); additional MAs were identified by cytogenetic & fluorescent in situ hybridization analyses. Results: Median age was 8 years (range birth - 44 yrs). MAs were identified in 90.1% of patients & included a median of 2 mutations (range 0-18) in 133 cancer-related genes. In contrast to genomic analyses of de novo malignancies in children, a high frequency of TP53 MAs was identified (20.4% of patients) and was associated with inferior survival. MAs were identified in targetable pathways including cell cycle regulation (32.6%), DNA repair (7.2%), epigenetic (28.6%), RAS/RAF/MEK (24%), tyrosine kinase (TK; 18.4%), PI3K/AKT/mTOR (11.8%), and NOTCH/WNT (8.9%). A higher number of MAs was associated with inferior survival. Patients with alterations in epigenetic & TK pathways also had inferior outcomes. MAs were frequently independent of histology & the spectrum of mutations was similar to adult cancers. Exceptional responses were observed with MA-based assignment of therapies (epigenetic, NTRK, RAS/RAF/MEK & ALK). Conclusions: Relapsed/refractory pediatric & AYA cancers have frequent MAs independent of histology. The spectrum of MAs is distinct from de novo disease & potentially reflects tumor evolution & resistance mechanisms. These findings support MA-guided approaches to new drug development paired with adult trials.

Macrophage Activation Syndrome (MAS) is a form of secondary hemophagocytic lymphohistiocytosis (HLH) that frequently complicates various rheumatoid disorders. Similar to HLH, these disorders present with chronic inflammation and immune dysfunction. Most typically, an identifiable infection triggers an immune response that is either ineffective due to genetic inactivating mutations that affect lymphocyte cytotoxicity, as in primary HLH, or uncontrolled and characterized by expansion of activated T and macrophage cells as in the case of MAS. While lymphocyte function is generally thought to be a crucial aspect of the disease state, abnormal myeloid cell activation by genetic activation of inflammasomes or repeated TLR stimulation was recently shown to result in MAS. These studies have implicated several cytokines, including SCF, IL-3, and GM-CSF in the disease process. A lymphocyte-independent model could be useful to isolate the myeloid contribution to MAS and would allow the testing of additional therapeutic strategies that target the effector cells directly. Such novel therapies are needed to address refractory cases that often prove fatal. Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross species activities of SCF, IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to non-transgenontrols. After engraftment with UCB, these mice develop severe, fatal MAS characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias and evidence of chronic inflammation. The BM becomes strikingly hypocellular and spleens are significantly enlarged with evidence of extramedullary hematopoiesis. Activated macrophages engaged in hemophagocytosis are readily detectable in the spleen and liver. Mice also show elevated body temperature and sCD25, indicating the presence of activated lymphocytes. Treatment of mice with the CD52 inhibitor alemtuzumab eliminated the human graft and resolved MAS, implicating a human cell and demonstrating the reversibility of the phenotype. However, there was no response to lymphocyte suppressive therapies such as steroids, intravenous immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease, demonstrating the utility of targeting the effector macrophage cells directly in this model. Using a multiplex ELISA assay, TNF alpha and IFN gamma were low to absent in affected mice, however, IL-6, IL-10, MIP-1 alpha, and MIP-1 beta were consistently upregulated. IL-6, MIP-1 alpha, and MIP-1 beta were lost after gemtuzumab ozogamicin therapy, but not after combined B/T cell ablation with rituximab and OKT-3. Interestingly, IL-10 and sCD25 was suppressed by either treatment. Since the IL-6R antibody tocilizumab is currently being evaluated in a clinical trial for HLH, we examined the efficacy of tocilizumab injections in our model and found delayed progression of anemia and a prolonged lifespan for affected mice. This novel model of MAS provides a new tool for the investigation of the mechanisms driving MAS and for the testing of myeloid-directed therapies in a human cell model. Disclosures No relevant conflicts of interest to declare.

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

We report a novel glucose-6-phosphate dehydrogenase (G6PD) mutation, which we propose to name G6PD Cincinnati (c.1037A>T, p.N346I), found in combination with G6PD Gastonia (c.637G>T, p.V213L) in an infant who presented with neonatal cholestasis. The G6PD Cincinnati mutation results in a non-conservative amino acid substitution at the tetramer interface disturbing its formation, as seen by native gel electrophoresis and immunoblotting. G6PD Gastonia disrupts dimerization of the enzyme and by itself causes chronic non-spherocytic hemolytic anemia. The G6PD Cincinnati mutation may have aggravated the clinical picture of G6PD Gastonia with the result of severe perinatal hemolysis causing cholestasis and associated liver injury.

As acute myeloid leukemia (AML) xenograft models improve, the potential for using them to evaluate novel therapeutic strategies becomes more appealing. Currently, there is little information on using standard chemotherapy regimens in AML xenografts. Here we have characterized the immunodeficient mouse response to combined Ara-C (cytarabine) and doxorubicin treatment. We observed significant toxicity associated with doxorubicin that required optimization of the route of injection as well as the maximum-tolerated dose for immunodeficient strains. Mice treated with an optimized 5-day induction protocol showed transient weight loss, short-term reduction of peripheral blood cell and platelet counts, and slight anemia. Considerable cytotoxicity was observed in the bone marrow (BM), with primitive LSK cells having a significant survival advantage relative to more mature cells, consistent with the idea of chemotherapy targeting actively growing cells. Treated leukemic mice demonstrated reduced disease burden and increased survival, demonstrating efficacy. AML cells showed significantly increased sensitivity to doxorubicin-containing therapy compared with murine BM cells. Although early treatment could result in some cures, mice with significant leukemia grafts were not cured by using induction therapy alone. Overall, the data show that this model system is useful for the evaluation of novel chemotherapies in combination with standard induction therapy.

RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.

10541Background: CPX-351, a liposomal formulation of cytarabine and daunorubicin in a 5:1 molar ratio, has demonstrated pre-clinical activity against AML and ALL cell lines and is currently under p...

The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

One key limitation of hematopoietic xenograft mouse models is the relative lack of effective myelopoiesis compared with human hematopoiesis. NSGS mice expressing human myelo-supportive cytokines allow for improved myeloid cell output upon human hematopoietic cell transfer. Surprisingly, we found that NSGS mice developed a severe, eventually fatal anemia upon humanization with umbilical cord blood (UCB) CD34+ cells. Anemia was characterized by a consistent, progressive drop in red blood cell numbers and hemoglobin that was correlated with higher MCV and an increased reticulocyte production at early time points. Eventually total white blood cell and platelet counts were also affected and reticulocyte numbers declined to or below normal. These effects were only apparent in mice with human grafts, and were also not observed in NSG mice with human grafts. Bone marrow cellularity was 40% of control humanized NSG mice and spleens were significantly larger, implying bone marrow dysfunction and extra medullary hematopoiesis. Bone marrow sections from anemic mice clearly showed a general hypocellularity with abundant fatty tissue filling the majority of the intramedullary space. We were able to recover red blood cell counts and hemoglobin to some degree upon transfusion of non-humanized mouse blood, however, the effect lasted only 7 days. CFSE labeling of anemic blood allowed us to detect a decreased half-life of red blood cells of 20 days compared to 30 days for humanized NSG mice. Treatment of mice with Campath, a CD52 antibody, completely reversed the phenotype at the expense of the entire human graft, demonstrating clearly that this was a graft-mediated process. However, specific ablation of B and T cells with rituximab and OKT3 did not alter the phenotype, even when administered soon after CD34+ cell transplant to suppress development of lymphocytes. Additionally, dexamethasone and IV/IG were ineffective in reversing the disease. These data, coupled with negative findings from tests to detect mouse reactive human antibodies, point towards myeloid cell involvement. Indeed, further experiments revealed that specific ablation of human myeloid cells effectively reversed the anemia. In summary, these data highlight a potential pitfall for effective myelopoiesis in the xenograft setting. More broadly, these findings demonstrate the possibility for myeloid driven bone marrow dysfunction and may warrant investigation of myeloid cells in cases of idiopathic aplastic anemia that fail to respond to therapies directed at autoimmune lymphocyte activity. Disclosures: No relevant conflicts of interest to declare.