ALBERT

Description: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.

Description: Introduction: Identification of tumor associated antigens (TAA) in acute myeloid leukemia (AML) will facilitate the use of targeted immunotherapies such as chimeric antigen receptor T cell therapy and bispecific antibodies. The characteristics of an ideal TAA include tumor specificity, high expression level, large percentage of positive tumor cells, and evidence for expression on putative cancer stem cells. V-domain Ig Suppressor of T cell Activation (VISTA) is a cell surface protein previously shown to be present on hematologic cells and inhibitory towards T cells. VISTA's role and presence within AML has yet to be defined. We performed detailed studies to characterize VISTA expression on tumor cells and mononuclear cell subsets in patients with AML. Methods: Immunophenotyping was performed on mononuclear cells from primary AML patient specimens using mass cytometry (CyTOF) by staining samples with a panel containing 34 antibodies to surface markers and cytokines (Table 1). This antibody panel was used to identify blast cells (variable immunophenotype), leukemic stem cells (LSCs) (CD34+, CD38-), T cells (CD3+), monocytes (CD33+, CD14+, CD11b+, HLA-DR+), myeloid derived suppressor cells (MDSCs) (CD33+, CD11b+, HLA-DR-), and M2 macrophages (CD11b+, CD163+). Raw median VISTA expression and percentage of cells expressing VISTA were calculated for all of these cell types. Samples were split into 2 categories based on blast VISTA expression. A sample was considered VISTA high if 〉10% of the blasts expressed VISTA, and VISTA low if

Description: Introduction: Acute myeloid leukemia (AML) is the most common blood cancer in adults and the second most common in children. Despite intensification of chemotherapeutic regimens, survival rates have plateaued. AML is therefore a candidate for novel therapeutics, specifically immune checkpoint inhibitors (ICIs). ICIs have shown significant promise in solid malignancies and more recently in Hodgkin's disease. However, it is currently not possible to predict which diseases and/or patients will benefit from ICIs. We are therefore, performing detailed studies to characterize the phenotype such that patients can be matched with ICI regimens to which they are most likely to benefit. Specifically, we are identifying the relevant lymphoid cell subsets present in the marrow of patients with AML, evaluating their differentiation and activation status, and quantifying the density of immune modifiers on cells of interest. We are also performing functional assays to further define the status of T cells in the AML microenvironment by evaluating the responsiveness of these cells to stimulation. Additionally, these studies assess the impact of ICI on the proliferative and cytokine production capacity of T cells and tumor cells resident in AML bone marrow. Methods: Immunophenotyping was performed on mononuclear cells from primary AML patient specimens using mass cytometry by staining samples with a panel containing 32 antibodies to surface markers and cytokines (Table 1). Each antibody was tagged with a unique lanthanide isotope and data was obtained using a DVS time-of-flight mass cytometer (CyTOF). T cells were defined by CD45 and CD3 positivity and then further differentiated based on the presence of CD4 or CD8. CD4 and CD8 cells were further classified into naive (CCR7+, CD45RA+), central memory (CCR7+, CD45RA-), effector memory (CCR7-, CD45RA-), and effector memory CD45RA+ (TEMRA)(CCR7-, CD45RA+) cells. Functional assays measured both the proliferative capacity and cytokine production profile of bone marrow T cells in response to CD3 stimulation in the context of the tumor microenvironment. Changes to the baseline T cell functional profile in the presence of various ICIs, including antibodies against PD1, CTLA4, TIM3, and VISTA, were measured. Results: Bone marrow samples from 9 patients with AML were collected. T cell percentages ranged from 0.2% to 4.3% with an average CD4:CD8 ratio of 0.39 to 2.0 (Figure 1a). Within the CD8 T cell population, naive, central memory, effector memory and TEMRA cells ranged from 1.1% to 25.4%, 1.8% to 9.7%, 15.5% to 51.8% and 15.5% to 51.8%, respectively (Figure 1b). Within the CD4 T cell population, naive, central memory, effector memory T cells and TEMRA cells ranged from 3.5% to 40.9%, 13.6% to 53.4%, 17.9% to 58.5%, and 0% to 10.2% respectively (Figure 1c). T cell expression of immune modulators varied (Figure 2) and there were differential effects of several ICIs on T cell proliferation and production of cytokines, including interferon gamma, interleukin-6, and tumor necrosis factor-alpha. Conclusion: These results provide the beginning of a functional and phenotypic description of the immune status in the marrow of patients with AML. Our results show that, like the disease itself, the immune microenvironment is very heterogeneous between samples with differential responses to ICIs suggesting that certain subsets of AML may be potential candidates for immunotherapy. This is a report of initial findings from our study; accrual of patient samples is ongoing to expand the power of our observations. Disclosures Huang: Janssen Pharmaceuticals: Employment. Sasser:Janssen Pharmaceuticals: Employment. Tyner:Aptose Biosciences: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding. Lind:Janssen Pharmaceuticals: Research Funding.

Description: Introduction: Blinatumomab is a bispecific antibody approved for the use in adults with relapsed acute lymphoblastic leukemia (ALL) but only about 50% of patients respond. A few brief reports have suggested that differences in T cell subsets between responders and non-responders may contribute to the failure of blinatumomab, however our understanding of the immune microenvironment in ALL is still in its infancy. We hypothesize response may be related to an immunosuppressive T cell microenvironment and in this project sought to define this microenvironment using both phenotypic and functional assays. Methods: Peripheral blood or bone marrow aspirate samples were obtained from patients with newly diagnosed and relapsed/refractory ALL as well as healthy donors. Mononuclear cells (consisting of lymphocytes and leukemic cells) were isolated via ficoll hypaque gradient. Immunophenotyping was performed by staining samples with a panel of 33 lanthanide isotope labeled antibodies and analyzing via CyTOF (Table 1). In parallel, functional assays were performed by labeling mononuclear cells with Cell Trace Violet and incubating on plates coated with an anti-CD3 antibody for 5 days. T cell division was assessed by flow cytometry. A portion of samples were cultured with blinatumomab 10 ng for 5 days and T cell division and the number of CD19+ cells were enumerated. Limited phenotyping and expression of checkpoint molecules after stimulation with CD3 or blinatumomab was assessed using multi-parameter flow cytometry for the following cell surface markers: CD45, CD3, CD4, CD8, CD56, CD19; and the checkpoint molecules PD-1, PL-L1, PD-L2, TIGIT, TIM-3, and CTLA4. Results: Bone marrow aspirate from 15 patients and PBMC from 4 patients with ALL were collected from patients with de novo (9), relapsed (7) or treatment refractory ALL (3). Bone marrow aspirate from 4 healthy donors were obtained as controls. The CD4:CD8 ratio in the healthy patient samples averaged 1.65. and was similar but more varied in the newly diagnosed patient samples. There was a marked expansion of CD8+ cells in the relapsed patient samples, while in the patients who had been treated but had residual disease, there was a marked reduction in CD8+ cells (figure 1). There was a trend to a higher average number of T regs in the patient samples than in the controls (6.02 vs 9.1). Within the CD8 T cell population, there was a trend toward higher numbers of effector memory (EM) and terminally differentiated T cells (TERMA) in the patient samples compared to the healthy donors (figure 2). CD57 was upregulated in the patient samples, and expressed primarily on the EM and TEMRA CD8+ cells. Expression of immune modulators on T cells was variable across the patient samples, with PD-1, TIGIT, and ICOS being the most highly expressed. Commonly expressed immune modulators on the leukemic blasts included LAG-3, CD112, 41BB and PD-L2. In terms of functional status, T cells from the patient samples did not proliferate in any of the 10 samples tested. This is in contrast to healthy donors where T cells divide in all instances. Six of 8 patient samples showed vigorous T cell division in response to culture with blinatumomab, with a marked reduction in CD19+ cells noted in the responders. Following blinatumomab stimulation, T cells demonstrated upregulation of PD-1 and TIGIT, while no changes were observed in the expression of TIM-3, CTLA-4 or PD-L1/2. Conclusion: These results highlight the differences in the T cell subsets and function between healthy donors and patients with acute lymphoblastic leukemia. ALL tends to lead to an expansion of CD8+ cells within the bone marrow, with a higher proportion of EM and TEMRA CD8+ cells as compared to healthy controls. High expression of CD57 was found on the EM and TERMA CD8+ T cell subsets of patients; while this is a well known marker of senescence it may also represent T cell exhaustion in this population. The most commonly upregulated checkpoints included TIGIT and PD-1 on T cells as well as the TIGIT ligand, CD112, on the tumor, suggesting that these axes may be targetable in ALL. The lack of response to CD3 stimulation in the patient samples suggests that the microenvironment in ALL is highly immunosuppressive. At this time patient numbers are small, however these results provide the beginning of a functional and phenotypic description of T cell subsets in the marrow and periphery of patients with ALL. Disclosures Leonard: Amgen: Research Funding. Druker:Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; GRAIL: 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; Patient True Talk: Consultancy; Millipore: Patents & Royalties; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Novartis Pharmaceuticals: Research Funding; Bristol-Meyers Squibb: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties. Tyner:Gilead: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Takeda: Research Funding; Constellation: Research Funding; Genentech: Research Funding; AstraZeneca: Research Funding. Lind:Fluidigm: Honoraria; Amgen: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Monojul: Research Funding.

Description: To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5–conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.

Description: Introduction: Immune checkpoint inhibitors (ICIs) have shown significant efficacy in solid malignancies and recently in Hodgkin's disease. However, there are presently no biomarkers that predict which cancer types and/or individual patients will benefit from ICIs. Despite intensification of chemotherapeutic regimens, survival rates in acute myeloid leukemia (AML) have plateaued. We are therefore, performing studies to characterize the immune phenotype and genetic profile of AML patients who may respond to ICI therapy. Specifically, we are identifying cell subsets present in the marrow of patients with AML, evaluating their differentiation and activation status, and quantifying the density of immune modifiers. We are also performing functional assays to define the status of T cells in the AML microenvironment by evaluating the responsiveness of these cells to stimulation. Additionally, these studies assess the impact of several ICIs on the proliferative potential and cytokine production capacity of T cells resident in the bone marrow of AML patients. Methods: Immunophenotyping was performed on the diagnostic bone marrow (Dx BM), diagnostic peripheral blood (Dx PB), and remission bone marrow (CR BM) for a single patient with AML. These studies used a time-of-flight mass cytometer (CyTOF) by staining samples with a panel containing 35 antibodies to surface markers and cytokines (Table 1). T cells were defined and then differentiated based on the presence of CD3 and CD4 or CD8. T cells were further classified into naive, central memory (CM), effector memory (EM), and effector cells. Functional assays measured proliferative capacity and cytokine production profile of bone marrow T cells in response to CD3 stimulation for the Dx BM and Dx PB. Changes to the baseline T cell functional profile in the presence of antibodies against PD1, CTLA4, TIM3, and TIGIT were measured in the Dx BM. Results: T cell percentages were 3.14%, 5.46%, and 17.1% for the Dx BM, Dx, PB, and CR BM, respectively. CD4:CD8 ratios were 3.5, 2.9, and 4.5, respectively. EM cells made up the largest percentage of CD8+ T cells in the Dx BM (40.99%), Dx PB (57.06%), and CR BM (53.55%). Naïve (52.49%), EM (37.63%) and CM (44.3%) cells made up the largest percentages of CD4+ T cells in the Dx BM, Dx PB, and CR BM, respectively (Figure 1). Checkpoint expression was absent (median intensity 〈 1) on all T cells except for some CD8+ subsets (Figure 2). Within the Dx BM, the effector (1.93) and EM (4.34) cells expressed TIGIT and the EM cells expressed PD1 (1.92). Within the Dx PB, the effector (1.81) and EM (4.0) cells expressed TIGIT. Within the CR BM, the effector cells (2.3) expressed TIGIT. In response to CD3 stimulation, 57.5% of the Dx PB T cells divided while there was no proliferation observed in the Dx BM. In response to CD3 stimulation and PD1 blockade, 28.2% of the Dx BM T cells divided and there was a 4-fold increase in IFNγ production. There was no division in the Dx BM T cells in response to TIGIT blockade but there was a 10-fold and 121-fold increase in IL-2 and IFNγ production, respectively. There was no significant proliferation or cytokine production in response to TIM3 or CTLA4 blockade. Conclusion: We observed differential T cell responses to stimulation in the presence of ICIs in this patient. The Dx PB T cells were capable of proliferating in the presence of CD3 stimulation alone whereas the Dx BM T cells were only able to divide in the presence of PD1 blocking antibody. PD1 blockade elicited proliferation and low levels of IFNγ production whereas TIGIT blockade elicited negligible proliferation but high levels of IFNγ production. There was low but differential checkpoint expression on T cells, with PD1 and TIGIT having the highest expression, specifically on CD8+ T cell subsets. The predominant CD8+ T cell subset, along with differential checkpoint expression, persisted into the CR BM but there was a shift in the predominant CD4+ T cell subset. These data suggest the presence of immune suppression in the bone marrow microenvironment that may be amenable to checkpoint inhibition. Disclosures Huang: Janssen Research & Development, LLC: Employment, Equity Ownership. Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

Description: Blinatumomab is currently approved for use as a single agent in relapsed and refractory Acute Lymphoblastic Leukemia. Cytotoxicity is mediated via signaling through the T-cell Receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia Chromosome positive ALL (Ph+ALL). However, some second and third generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual ABL/Src inhibitors with blinatumomab in vitro from both healthy donor samples as well as in primary samples from patients with Ph+ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells, as well as enhanced production of IFN-gamma. The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation nor IFN-gamma production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-gamma production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples as well as samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies highlighting the importance of maintaining T-cell function with targeted therapies.