Abstract: Major efforts are under way to develop combination therapies to target multiple biologic pathways for effective synergistic cell killing and decrease the risk of cancer relapse. However, a major technical challenge is the lack of screening platforms that allow assessment of optimal combinations directly in individuals. Flow cytometry is a widely used option; however, the large amount of sample required limits the number of drug combinations that can be tested on primary patient samples. Moreover, many protein targets, especially intracellular proteins (e.g., phosphoproteins, immune modulatory molecules) are often present at low levels, making it challenging to detect via flow cytometry or other means—especially in conditions of drug inhibition whereby signal cannot be conclusively discriminated from background noise. We have developed a next-gen miniaturized single-cell imaging platform that evaluates the effect of drug combinations in primary patient tumor and immune cells, with quantitative detection sensitivity and single-cell granularity. We demonstrate the use of this platform technology to screen interactions between targeted agents and immune checkpoint inhibitors (ICIs) in individuals with acute myeloid leukemia (AML). In many tumor types, including AML, targeting tumor cells with small-molecule drugs while concomitantly inducing an antitumor immune response has the possibility of synergistic activities that avoid therapeutic resistance (1). However, many of the pathways of proliferation and survival that are targeted with small-molecule drugs are also important for the ability of tumor-reactive T cells to expand and function (2). For this reason, there is a distinct possibility that many drugs designed to kill tumor cells will also impair T-cell responses and thus not be compatible with immunotherapies such as ICIs. We show how platform functional readouts of ex vivo T-cell activation and tumor-cell killing, along with conventional and machine learning image-based single-cell analysis, provide new information on the effect of specific combination/single agents in individuals. We report observations of ICI rescue of T-cell proliferation and the synergistic effects of TIM3, MEK, and other combination agents. These results demonstrate the advantages of this precision technology to obtain new functional information that helps identify promising combinations—and to do so directly on samples that represent the functional and genetic diversity seen in AML (3). References: 1. Hughes PE et al. Targeted therapy and checkpoint immunotherapy combinations for the treatment of cancer. Trends in Immunotherapy 2016. 2. Zitvogel L et al. Immunological aspect of cancer chemotherapy. Nature Reviews Immunology 2008. 3. Tyner JW et al. Functional genomic landscape of acute myeloid leukaemia. Nature 2018. Note: This abstract was not presented at the conference. Citation Format: Thomas Jacob, Yunqi Yan, Yoko Kosaka, Sophia Jena, Stephen E. Kurtz, Andy Kaempf, Tomi Mori, Young Hwan Chang, Bill H. Chang, Uma Borate, Elie Traer, Shannon K. McWeeney, Jody Martin, Jeffrey W. Tyner, Evan F. Lind, Tania Q. Vu. Advancing precision medicine combination drug screening: A miniaturized single-cell imaging platform for evaluating immunotherapy-small molecule combination therapeutics in individuals [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 08.

Abstract: Ex vivo drug profiling captures disease-relevant features and relevant sensitivity to therapeutic agents in ALL. A subset of drug-resistant T-ALL without mutations in ABL1 is highly responsive to dasatinib, which provides a rationale for drug repurposing.

Abstract: DNA replication timing of 〉 100 pediatric leukemic samples identified BCP-ALL subtype-specific genome alteration signatures. Comparative analyses identified features of specific stages of B-cell differentiation and potential associations with clinical outcome.

Abstract: Kinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of kinase targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options. Cancer Res; 73(1); 285–96. ©2012 AACR.

Abstract: 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: Background We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients (Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutations (the most common of which is T618I) and truncation mutations. Drug and siRNA screening of primary patient samples revealed that the two classes of CSF3R mutations exhibit differential sensitivity to inhibition of SRC or JAK kinases. CSF3R truncation mutations conferred sensitivity to SRC family kinase inhibition, while CSF3R membrane proximal mutations (T618I) conferred sensitivity to JAK kinase inhibition. A patient with the T618I membrane proximal mutation responded to treatment with the FDA approved JAK inhibitor, ruxolitinib. CSF3R truncation mutations have also been observed in a subset of severe congenital neutropenia patients who are at high risk for development of acute myeloid leukemia. Prior studies in this context have shown that truncation mutations result in loss of endocytic and degradation motifs, leading to increased expression of the receptor. The differences in signaling and drug sensitivity of these mutation classes suggest that membrane proximal mutations may activate CSF3R signaling through a distinct, as-yet unknown mechanism. Furthermore, a subset of CNL patients harbor both membrane proximal and truncation mutations on the same allele, though the consequences of these compound mutations are not yet known. Methods CSF3R expression level and banding pattern were assessed by immunoblot of lysates from 293T17 cells transfected with wild type, membrane proximal mutant, or truncation mutant CSF3R. O-linked glycosylation was removed from the receptor by treatment with O-glycosidase and neuraminidase. Ligand independence of the CSF3R mutants was analyzed in murine interleukin-3 (IL3)-dependent Ba/F3 cells. CSF3R dimerization was assessed by co-transfecting CSF3R-Flag and CSF3R-V5 tagged constructs and then immunoprecipitating CSF3R-Flag and detecting co-immunoprecipitation of the CSF3R-V5 by immunoblot. Transforming potential of the CSF3R compound mutations relative to the corresponding point or truncation mutations was assessed by analyzing IL3-independent growth of Ba/F3 cells or mouse bone marrow colony formation. Results To better understand the functional and biochemical differences between membrane proximal and truncation mutant CSF3R, we examined transformation potential, requirement for ligand, and expression patterns in Ba/F3 and 293T17 cells. We found membrane proximal mutations to exhibit rapid transformation potential and ligand independence, while truncation mutations exhibited delayed transformation and ligand hypersensitivity. Unlike the truncation mutations, which induce dramatic overexpression of CSF3R, the T618I mutation did not result in overexpression of the receptor but instead induced a shifted banding pattern, indicative of altered protein modification. We examined the amino acid sequence surrounding the membrane proximal mutations and found residue T618 to be part of a consensus motif for O-glycosylation, wherein wild type CSF3R is O-glycosylated and the T618I mutation abrogates this O-glycosylation event. Furthermore, the T618I mutation exhibited increased receptor dimerization compared to wild type CSF3R, which likely explains its ligand independence. Finally, we found that CSF3R compound mutations have increased transforming potential in Ba/F3 and murine bone marrow colony assays compared with either class of single mutation, further underscoring the different mechanisms of action of the membrane proximal and truncation mutations. Conclusion CSF3R represents a promising therapeutic target for patients with CNL. We show that T618I, the most common CSF3R mutation in CNL, is part of an O-linked glycosylation site. Mutation of this residue leads to loss of O-linked glycosylation and represents a novel mechanism of homodimeric cytokine receptor activation. CSF3R compound mutations are more rapidly transforming relative to the membrane proximal or truncation mutations alone, warranting their further investigation for patient prognosis and therapy. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Gotlib:Incyte: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel Support Other. Fleischman:Incyte: Speakers Bureau. Collins:Genoptix: Membership on an entity’s Board of Directors or advisory committees. Oh:Incyte Corporation: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Deininger:Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding. Druker:Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other. Tyner:Incyte Corporation: Research Funding.