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Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.

PDF file, 421K, Supplementary Figure 1: Specificity of imatinib, dasatinib, PPY-A, BAW667, and a SCF-blocking antibody on Mo7ep210BCR-ABL1 cells. Supplementary Figure 2: Comparison of CFU-GM colony formation upon sole BCR-ABL1 inhibition (PPY-A), sole KIT inhibition or dual BCR-ABL1/KIT inhibition. Supplementary Figure 3: Inhibition of KIT in human cells through use of a lentiviral vector for simultaneous expression of shKIT and GFP . Supplementary Figure 4: Analysis of CML CFU-GM colony growth following removal of the individual cytokines. Supplementary Figure 5: BCR-ABL1 and KIT signaling influence activation of AKT associated with a reduction of Foxo3A. Supplementary Table 1. Activity profile of BAW667.

Background: Multiple myeloma (MM) is an incurable disease with a heterogenous clinical course and genomic landscape. Autologous anti-BCMA chimeric antigen receptor (CAR) T-cells are a promising new therapy, but determinants of response and resistance are not well known. Cell-free DNA (cfDNA) is a useful tool to study MM as it allows for repeated, non-invasive tumor assessment. We apply a novel method for simultaneously tracking tumor mutations and CAR T-cells from cfDNA using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). Methods: We designed a 480kb CAPP-Seq hybrid capture panel to identify mutations, track tumor burden and minimal residual disease, and detect cfDNA derived from the CAR transgene (CAR-cfDNA) in patients receiving idecabtagene vicleucel (ide-cel). Flow cytometry (FC) for enumeration of CAR T-cells was performed from peripheral blood mononuclear cells (PBMCs) when available. Results: We profiled 153 biologic samples, including plasma, PBMCs, and bone marrow mononuclear cells, from 15 patients receiving ide-cel and 18 healthy controls. We observed a median of 84 SNVs (range 30-277) prior to therapy. Patients with prolonged responses (>90 days) had significantly lower circulating tumor DNA (ctDNA) at day 28 post-infusion than patients with early progression ( Conclusions: Cell-free DNA is a promising biomarker for mutational genotyping, disease monitoring, and tracking CAR T-cells in MM. The persistence of CAR-cfDNA has particular prognostic importance and novel strategies to increase CAR persistence should be explored. Citation Format: Mia Carleton, Hitomi Hosoya, Kailee L. Tanaka, Brian Sworder, Vanna Hovanky, Bita Sahaf, Matthew J. Frank, George E. Duran, Tian Y. Zhang, Sally Arai, David Iberri, Michaela Liedtke, David B. Miklos, Michael S. Khodadoust, Surbhi Sidana, David M. Kurtz. Tumor and immune determinants of response to anti-BCMA CAR T-cell therapy in multiple myeloma using cell-free DNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5707.

As a therapeutic modality, cancer vaccination leverages the well-characterized ability of antigen presenting cells (APCs) to take up and present tumor antigens in an effort to stimulate potent anti-cancer T cell responses. However, clinical successes with therapeutic cancer vaccination remains limited. Here, we present a novel cancer vaccination approach utilizing myeloid lineage reprogramming to directly convert cancer cells into tumor reprogramed-APCs (TR-APCs).In order to understand the therapeutic potential of TR-APCs, we generated syngeneic murine B-cell acute lymphoblastic leukemia models amenable to myeloid lineage reprogramming via ectopic expression of the myeloid lineage transcription factors PU.1 (Spi1) and C/EBPα (CEBPA). Upon enforced expression of these factors, the resulting TR-APCs acquire both myeloid phenotype and function, including the capacity for phagocytosis and antigen presentation. Crucially, TR-APCs can present endogenous self-derived tumor antigens directly encoded in their genome, without the need for phagocytosis and processing of adjacent tumor cells. In vitro, leukemia-derived TR-APCs express enhanced levels of antigen presentation machinery and co-stimulatory molecules, and potently stimulate tumor-specific CD4+ and CD8+ T cells. While in vivo TR-APC induction elicits only a modest extension to overall survival in immunodeficient hosts, generation of TR-APCs in immunocompetent syngeneic animals leads to complete leukemia eradication and protection from subsequent re-challenge. Further analysis of the in vivo immunological response to TR-APC induction revealed oligoclonal T cell expansion and establishment of cancer-specific immunological memory. Strikingly, use of a dual flank tumor model revealed that local TR-APC induction is sufficient to elicit systemic immunity capable of eradicating distant metastatic sites. We extended this treatment modality beyond hematologic malignancies, demonstrating that some solid tumors, including sarcomas and carcinomas, are amenable to myeloid-lineage reprogramming into TR-APCs, and contribute to increased overall survival. Finally, we demonstrate the clinical applicability of this approach by generating TR-APCs from primary human B cell acute lymphoblastic leukemia (B-ALL) specimens. Strikingly, primary B-ALL-derived TR-APCs stimulate autologous patient-derived T cells, demonstrating the clinical potential of TR-APCs to enhance antitumor immunity in patients. Thus, TR-APCs represent a novel cancer vaccination therapeutic strategy with broad implications for clinical immuno-oncology. Citation Format: Miles H. Linde, Sarah F. Gurev, Paul Phan, Feifei Zhao, Eric J. Gars, Melissa Stafford, Thomas Köhnke, Payton L. Marshall, Amy C. Fan, Christopher G. Dove, Ian L. Linde, Lindsay P. Miller, Robbie G. Majzner, Tian Yi Zhang, Ravindra Majeti. Reprogramming cancer into antigen presenting cells as a novel immunotherapy [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 1505.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.