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Acute Lymphoblastic Leukemia (ALL) harboring the t(17;19)(q22;p13) is a rare subtype of leukemia with a dismal prognosis. This recurring translocation produces an aberrant TCF3-HLF fusion with distinct gene expression profiles and drug sensitivity. Recent studies have shown that this subtype of ALL might be targeted using therapies inhibiting BCL-2 and the pre-B cell receptor through inhibition of SRC family kinases. However, preliminary validation of these studies have revealed significant heterogeneity of response to BCL-2 and SRC inhibitors. As such, we sought to identify other possible targets that could overcome this heterogeneity and improve response to therapy. Methods: One local as well as four other samples from the Children's Oncology Group's ALL Biorepository with TCF3-HLF ALL were expanded in immunodeficient NSG mice. All samples were verified by RT-PCR and Sanger sequencing for the fusion transcript. Samples were then interrogated with our functional drug screen that is comprised of compounds with activity against two-thirds of the tyrosine kinome as well as other non-tyrosine kinase pathways, including RAF/MEK/MAPKs, PI3K/AKT/mTOR, AMPK, ATM, Aurora kinases, CAMKs, CDKs, GSK3a/b, IKK, PKA, PKC, PLK1, and RAF as well as BCL2 family, BRD4, IDH1/2, Hedgehog, HSP90, NOTCH/g-secretase, proteasome, survivin, STAT3, and WNT/b-catenin. The samples were sequenced using the Agilent SureSelect Strand-Specific RNA Library Preparation Kit on the Bravo robot (Agilent). All five patient samples successfully engrafted into NSG mice and were tested for in vivo sensitivity as assessed for disease burden or survival. Results: Three patient samples were identified to carry Type I translocations fusing exon 13 of TCF3 with variable intronic insertions followed by exon 4 of HLF. All three type I translocations produced different fusions due to different lengths within the variable region. One sample predicted a truncation product of TCF3 ending in exon 13 with an early stop codon within the variable region. Two patient samples carried the identical type II translocation fusing Exon 12 of TCF3 with exon 4 of HLF. RNA-seq results of the five samples identified other individual translocations, but none involved other specific disease related lesions. Results from our drug screen showed significant heterogeneity in response to the majority of drugs assayed including the ABL/SRC inhibitor dasatinib and the BCL-2 inhibitor venetoclax. Further, in vivo studies exposing cohorts of animals to vehicle (n=5), dasatinib (40mg/kg/day; n=5), venetoclax (25-100mg/kg/day; n=5) or combination of dasatinib and venetoclax (n=5) identified only two samples with treatment benefit. Interestingly, review of the results of the drug screen suggested hypersensitivity to aurora kinase inhibitors. Each sample was tested in vivo in cohorts of vehicle (n=5) and alisertib (30mg/kg/day; n=5). All five ALL samples showed significant response (p Conclusion: Our results suggest that TCF3-HLF ALL is a heterogeneous subset of ALL with both different gene expression patterns from TCF3-HLF to other fusions as well as functional drug response. In vivo validation in the murine model with these five samples suggests significant heterogeneity to current pursued targets such as BCL-2 and SRC compared to previously published reports. Most intriguing, all samples tested with alisertib identified significant in vivo response suggesting unique preclinical support to pursue further clinical testing within this rare and lethal subtype of ALL. Disclosures Leonard: Amgen: Research Funding. Mullighan:Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy. Tyner:Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding. Druker:GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Patient True Talk: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; ARIAD: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Celgene: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties.

Transcription Factor 3 (TCF3) rearrangements are a recurring chromosomal abnormality in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in approximately five percent of pediatric ALL. Historically, the majority of these patients carried a poor prognosis, but advances with more intensive cytotoxic chemotherapy have improved the survival rate while exposing patients to increased short and long-term toxicities. Two genetic rearrangements produce the chimeric transcription factors, TCF3-PBX1 t(1;19)(q23;p13) and a much rarer TCF3-HLF t(17;19)(q22;p13). Sadly, TCF3-HLF remains an extremely difficult disease to treat with few, if any known survivors. Although it is unknown how these translocations lead directly to disease, it is established that they do result in diseases arrested in a later stage of B-cell differentiation and pre-B cell receptor (pre-BCR) dependence. Recently, we highlighted the concept of targeting the pre-BCR pathway for therapeutic potential using dasatinib (Sprycel). Here, we further examine dasatinib effectiveness in the murine xenograft model for TCF3-rearranged ALL. Methods: Primary patient samples were obtained with written informed consent approved by the Institutional Review Board of Oregon Health and Science University and processed. Mononuclear cells were separated by Ficoll and exposed to increasing concentrations of dasatinib. Inhibitory Concentration of fifty-percent viability (IC50) was calculated for each sample. The median IC50 for over four hundred acute leukemic samples interrogated by this assay was calculated to approximately 100nM. Samples with IC50 values below 30nM were deemed hypersensitive to dasatinib. For xenografts, frozen viable primary patient samples were thawed and grafted via tail-vein into NOD/SCID/IL-2rgnull(NSG) mice 24 hours after sub-lethal irradiation with 200 cGy. Upon engraftment, and in vivo expansion, animals were euthanized and leukemic cells recovered from the spleen were then injected in secondary recipients. One week after injection the mice were divided into two groups and treated by oral gavage with dasatinib at 50mg/kg/dose daily or citrate control for 5 days per week. Treatment continued until the day of sacrifice (4-20 weeks). Peripheral blood engraftment was monitored weekly starting on week 3 by flow cytometry analysis using anti-human CD19 and CD45 (hCD19-APC, hCD45-FITC) versus anti-murine CD45 (mCD45-PerCP-Cy5.5). Flow cytometric data was analyzed using FACS/AriaIII. Results: Screening over one hundred BCP-ALL samples identified that approximately ten percent of these samples show hypersensitivity to dasatinib. TCF3-rearranged ALL and BCR-ABL1 ALL had a majority of samples with IC50's less than 10nM. Throughout all known subsets of ALL except ETV6-RUNX1, there also appeared to be individual samples that have IC50 values less than 30nM, suggesting significant sensitivity to this drug. Of these, three individual TCF3-rearranged ALL samples were identified and xenografted into NSG mice, expanded and injected into secondary recipients. All dasatinib treated cohorts showed significantly less leukemic peripheral blood chimerisms as compared to their vehicle control counterparts. Further, in vitro treatment of xenografted cells with dasatinib indicated inhibition of the pre-BCR by decrease in pan-phospho-SRC. Intriguingly, dasatinib did not completely abolish disease in all TCF3-rearranged ALL, suggesting other important mechanisms for cell viability. Conclusions: These studies show in vivo therapeutic benefits of dasatinib as treatment for TCF3-rearranged ALL, and open the possibility of adding this drug to their treatment. Further studies are underway to address the mechanisms of dasatinib sensitivity of other subsets of ALL identified in our screen in hopes of adding targeted therapies to their treatment. Figure 1 Figure 1. Disclosures Druker: Molecular MD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other. Off Label Use: Dasatinib use as potential therapy in ALL.

Abstract 1378 Cell membrane-derived vesicles carrying protein and RNA cargo have recently emerged as potent mediators of cell-cell communication. These vesicles traffic from their tissue of origin into the blood stream, providing a serum-based and minimally invasive biomarker platform for cancer-specific RNA and protein signatures. We were interested in evaluating vesicle production by acute myeloid leukemia (AML) cells, with an emphasis on RNA content and cell-cell trafficking. Here we demonstrate for the first time that cell lines and primary cells from AML patients release exosome-sized vesicles (30–100nm) capable of transfer to bystander cells. Analysis of their non-protein content revealed the enrichment of many coding- and non-coding RNAs with relevance to AML biology, including prognostically important transcripts encoding FLT3, NPM-1, IGF-IR, CXCR4 and MMP9. In addition, we demonstrated the presence of a diverse group of transcription factor mRNAs (MYC, MEF2C, GATA1, SHIP1, ID1, E2F1, CEPBA CEPBB) in exosomes. PCR array studies also confirmed the broad representation of cellular miRNAs. Among the 234 miRNAs represented, the levels of several (miR −9, −155 and −223 and LET-7A) were enriched over the levels seen in donor cells (U6 RNA control). When examining cell-cell trafficking of AML exosomes, we noted rapid entry into co-cultured stromal cells, with maximum uptake occurring between 2 and 3 hours. AML-derived mRNA transcripts were transferred to bystander cells without direct cell-cell contact after Transwell co-culture or direct exposure to purified exosome preparations, or via vesicle-rich media. Results show that exosome trafficking induced changes in gene expression (OPN, ANGPT, SCF, SDF-1A) and altered the secretion of cytokines and angiopoietic growth factors by bone marrow stromal cells. Additional proof-of-concept studies provided evidence for the canonical function of transferred RNA. Specifically, we found that transfer of IGF-IR mRNA led to proliferative responses in co-cultured OP9 stromal cells that were abrogated by the IGF-IR specific inhibitor picropodophyllin. Further, exosomes derived from AML cell lines (HL60 and Molm14) altered c-FOS and VEGF expression in OP9 cells as well as improving viability and promoting migration toward SDF-1A by a hematopoietic progenitor cell line (Baf3), respectively. Our observations support the concept that AML exosomes exhibit regulatory potential in the leukemic bone marrow niche via direct cytoplasmic cell-cell transfer of transcripts encoding tyrosine kinase receptors and transcription factor as well as micro- RNAs. We propose that AML exosomes contribute to the extrinsic signaling cues in the microenvironment that promote disease resistance in AML and that they are rich in biomarkers to guide diagnosis and treatment. Disclosures: No relevant conflicts of interest to declare.

SummaryStudying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.