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The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. Significance: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov.

Supplemental Figure 1. Generation of a panel of iPSCs from patients with AML. Supplemental Figure 2. Reprogramming aids reconstruction of the evolutionary history and clonal composition of AML. Supplemental Figure 3. Transplantation of AML-iPSCs into immunodeficient mice. Supplemental Figure 4. Developmental block in a subset of AML-iPSC lines. Supplemental Figure 5. Transplantation of primary AML cells and patient-matched AMLiPSC lines. Supplemental Figure 6. Single-cell RNA-sequencing analyses of matched primary and iPSC-derived leukemia cells from patient AML-47. Supplemental Figure 7. Cell cycle and pseudotime analyses. Supplemental Figure 8. Comparison of scRNA-Seq data integration and clustering methods and pseudobulk differential gene expression analyses.

Table S1. Patient characteristics. AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; MPN: myeloproliferative neoplasm; ET: essential thrombocythemia; PBMCs: peripheral blood mononuclear cells; BMMCs: bone marrow mononuclear cells; PDX: patient-derived xenografts Table S2. All patient samples used in this study with genetic characterization and reprogramming outcomes. Blue font denotes partially reprogrammed (as opposed to bona fide iPSC) colonies and clones. Table S3. All AML-iPSC lines phenotypically characterized. Table S4. Top 50 upregulated genes (highest log2 fold change) in each cluster. Table S5. Primers used for genotyping. Table S6. Primers used for qRT-PCR analyses.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers.Significance:We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation.See related commentary by Doulatov.

Supplementary Table Legends 1-3, Methods, References from Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL. Cancer Res; 71(4); 1374–84. ©2010 AACR.

Supplementary Tables 1-3 from Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia

Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and "don't eat me" signal on cancer cells, that eliminates leukemia stem cells by inducing tumor phagocytosis. Hypomethylating agents synergize with magrolimab by inducing "eat me" signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab with azacitidine (AZA) has shown encouraging activity in frontline acute myeloid leukemia (AML) unfit for intensive chemotherapy and myelodysplastic syndrome. In TP53-mutant (TP53m) AML, it demonstrated an objective response rate of 69%, complete response (CR) rate of 45%, and median overall survival (OS) of 12.9 months. The TP53 gene mutation is observed in approximately 10-15% of newly diagnosed AML and is associated with poor survival. The published first-line median OS in TP53m AML is 5-7 months, regardless of whether patients are treated with intensive chemotherapy or non-intensive approaches, such as a hypomethylating agent with venetoclax (VEN). AML patients harboring the TP53 gene mutation represent a significant unmet medical need. Aims: To evaluate the efficacy, safety, and tolerability of magrolimab+AZA vs physician's choice of VEN+AZA or "7+3" chemotherapy in patients with previously untreated TP53m AML. Design and Methods: This is a phase 3, randomized, open-label, multicenter study. Approximately 346 patients will be randomized (1:1) to magrolimab+AZA (investigational arm) or physician's choice of VEN+AZA or 7+3 chemotherapy, based on patient fitness (NCT04778397). Randomization will be stratified by appropriateness for non-intensive vs intensive therapy, geographic region (US vs non-US sites), and age ( Status: Patients will be enrolled at approximately 140 centers globally. Accrual is ongoing. Disclosures Daver: Gilead Sciences, Inc.: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Glycomimetics: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Novimmune: Research Funding; Genentech: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Trovagene: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Trillium: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Vyas: Novartis: Honoraria; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Jazz: Honoraria; Janssen: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria; Takeda: Honoraria. Chao: Gilead Sciences, Inc.: Current Employment; TigaTx: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties; Hepatx Inc: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Iconovir Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Stanford University Medical School: Membership on an entity's Board of Directors or advisory committees; Foresite capital: Consultancy; Bioverge: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Chimera Bioengineering: Current equity holder in publicly-traded company. Xing: Gilead Sciences, Inc.: Current Employment. Renard: Gilead Sciences, Inc.: Current Employment, Current holder of individual stocks in a privately-held company; Alphabet: Current Employment, Current equity holder in publicly-traded company. Ramsingh: Gilead Sciences, Inc.: Current Employment, Current holder of individual stocks in a privately-held company. Sallman: Syndax: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta: Consultancy; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees. Wei: Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax: Patents & Royalties; MacroGenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche AG: Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Magrolimab is an investigational therapy

Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and "don't eat me" signal on cancer cells. Magrolimab induces tumor phagocytosis and eliminates leukemia stem cells. Chemotherapy and hypomethylating agents synergize with magrolimab by inducing "eat me" signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab+azacitidine (AZA) has shown encouraging clinical efficacy with an objective response rate (ORR) of 63% and a complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate of 54% in first-line acute myeloid leukemia (AML). Newly diagnosed AML patients (pts) who are ineligible for intensive chemotherapy (IC) are incurable despite the progress made with AZA+venetoclax (VEN; median overall survival [OS] 14-18 months), while AML pts with relapsed/refractory (R/R) disease after intensive regimens have a dismal prognosis. Furthermore, for pts in remission, maintenance therapy with oral AZA has improved OS and prevented relapse, although relapse rates remain high. Magrolimab combinations are being evaluated in each of these settings to improve the standard of care. Aims: To evaluate the safety, efficacy, and tolerability of magrolimab combined with antileukemia therapies in first-line pts ineligible for IC (cohort 1), R/R pts after IC (cohort 2), and pts in CR/CRi with presence of minimal residual disease (MRD) after IC (cohort 3). Design and Methods: This is an open-label, multiarm, multicenter study that includes 3 safety run-ins with corresponding phase 2 cohorts (NCT04778410). Pts in cohort 1 must be ≥75 years or 18-74 years with comorbidities that preclude IC. Pts in cohort 2 must have R/R AML after initial IC. Pts in cohort 3 must be ≥55 years and have achieved a CR/CRi with MRD positivity, as assessed by flow cytometry, after IC, and not be candidates for hematopoietic stem cell transplant. Each cohort will initially enroll 6 pts for 1 cycle (28 days) to assess dose-limiting toxicities and determine the recommended phase 2 dose (RP2D). Pts in cohort 1 will receive a combination of magrolimab, VEN, and AZA. Pts in cohort 2 will receive magrolimab in combination with mitoxantrone, etoposide, and cytarabine (MEC). Pts in cohort 3 will receive magrolimab with oral CC-486. All will receive magrolimab on the same schedule. In cycle 1, magrolimab will be administered intravenously (IV) as priming and ramp-up doses at 1 mg/kg on Days 1 and 4, 15 mg/kg on Day 8, and 30 mg/kg on Days 11, 15, and 22. In cycle 2, magrolimab 30 mg/kg will be administered weekly. From cycle 3 onward, magrolimab 60 mg/kg will be administered on Day 1. For cohort 1, AZA (75 mg/m 2) will be administered IV/subcutaneously on Days 1-7 or 1-5, 8, and 9 of each cycle. VEN, MEC, and CC-486 will be administered according to labeled indications. After completion of the safety run-in, additional pts will be enrolled in cohort 1 (n=40), cohort 2 (n=30), and cohort 3 (n=40) for phase 2. In phase 2, pts in cohorts 1 and 3 will receive study treatment at RP2D until disease progression, unacceptable toxicity, or loss of clinical benefit. Pts in cohort 2 will receive magrolimab with MEC for up to 3 cycles followed by magrolimab alone for a total of 12 magrolimab cycles; pts who do not achieve an objective response will discontinue after 2 cycles. In cohorts 1 and 2, the primary efficacy endpoint is the CR/CRi rate. Secondary endpoints of interest include OS, ORR, and MRD-negative CR/CRi rates. The primary efficacy endpoint in cohort 3 is the MRD-negative CR/CRi rate. Status: Accrual is ongoing. Disclosures Vyas: Novartis: Honoraria; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Gilead: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Daver: Abbvie: Consultancy, Research Funding; Glycomimetics: Research Funding; Trillium: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Hanmi: Research Funding; Novimmune: Research Funding; Genentech: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Pfizer: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Chao: Gilead Sciences, Inc.: Current Employment; TigaTx: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties; Hepatx Inc: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Iconovir Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Chimera Bioengineering: Current equity holder in publicly-traded company; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Bioverge: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Stanford University Medical School: Membership on an entity's Board of Directors or advisory committees; Foresite capital: Consultancy. Xing: Gilead Sciences, Inc.: Current Employment. Renard: Alphabet: Current Employment, Current equity holder in publicly-traded company; Gilead Sciences, Inc.: Current Employment, Current holder of individual stocks in a privately-held company. Ramsingh: Gilead Sciences, Inc.: Current Employment, Current holder of individual stocks in a privately-held company. Wei: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche AG: Research Funding; MacroGenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax: Patents & Royalties; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sallman: Takeda: Consultancy; Magenta: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Speakers Bureau. OffLabel Disclosure: Magrolimab is an investigational therapy

TPS7055 Background: MDS is a clonal myeloid disorder characterized by cytopenia and ineffective hematopoiesis. The median age of diagnosis is approximately 70 yrs of age and prognosis and treatment are guided by the Revised International Prognostic Scoring System (IPSS-R) criteria. Patients with intermediate, high and very high risk MDS (HR-MDS) have a median overall survival (OS) of 0.8 to 3.7 years. Despite the high unmet need in this patient population, azacitidine (AZA) is the only approved therapy for HR-MDS which has improved overall survival in clinical trials to date. However, these agents lead to low complete response (CR) rates (10-17%) with limited OS ( = 2 years), indicating a need for alternative therapy. Magrolimab is a first-in-class monoclonal antibody that blocks the macrophage inhibitory immune checkpoint CD47, a “do not eat me” signal overexpressed on tumor cells. Binding of magrolimab to CD47 leads to phagocytosis of tumor cells. AZA increases expression of prophagocytic “eat me” signals, facilitating synergy with magrolimab. In an ongoing phase 1b study, the combination of magrolimab + AZA led to high response rates (ORR 91%, with a CR of 42%) and an acceptable safety profile without significant immune-related adverse events. ENHANCE (NCT04313881) is a phase 3 trial comparing the efficacy and safety of magrolimab + AZA with that of AZA + placebo (PBO) in previously untreated patients with HR-MDS. Methods: Patients ≥18 years old with previously untreated intermediate to very high risk MDS by IPSS-R are eligible for ENHANCE. Randomization is 1:1 to magrolimab + AZA or AZA + PBO with no crossover allowed. Magrolimab or placebo is administered intravenously (IV) with an initial 1 mg/kg priming dose to mitigate on target anemia. An intrapatient dose escalation regimen up to 30 mg/kg is then administered through Cycle 1, 30 mg/kg weekly dosing in Cycle 2, with 30 mg/kg Q2W dosing occurring in Cycle 3 and beyond. AZA is administered per regional prescribing information. Patients may remain on treatment until disease progression, relapse, loss of clinical benefit, or until unacceptable toxicities occur. Two primary efficacy endpoints are CR rate and OS. For patients undergoing allogeneic stem cell transplantation (ASCT), data for the CR rate will be censored at the time of ASCT and OS will be censored at the last known alive date. Secondary efficacy endpoints include RBC transfusion independence rate, event-free survival, minimal residual disease-negative rate, time to AML transformation, and patient-reported Functional Assessment of Cancer Therapy (FACT)-Anemia response rate. Biomarkers of immune cell recruitment, immune cell signaling, and bone marrow penetration of magrolimab will also be explored. Planned enrollment is approximately 520 patients globally, which began in September 2020. Accrual is ongoing. Clinical trial information: NCT04313881.

The ability to epigenetically reprogram differentiated somatic cells to pluripotency resulting in the discovery of induced pluripotent stem cells (iPSCs), has unlocked fundamental biologic insights into numerous genetic diseases. These insights have resulted from the key property of iPSCs to differentiate into all cell lineages in an unlimited manner while maintaining the genetic identity of the originating cell. iPSCs have been utilized to investigate both monogenic and complex genetic disorders spanning hereditary and acquired diseases. Recently, iPSCs have been utilized to model human cancer, with a specific focus on modeling conditions of malignant hematopoiesis. In addition to serving as a genetic disease model in cancer, iPSCs can also be used as a tool to address a key question in interrogating the interaction between the cancer epigenome-genome. Specifically, how does reprogramming the epigenome affect cancer phenotype and specifically malignant hematopoiesis? This review will address this question and highlight the state of the field in generating iPSCs from hematologic malignancies, key biologic insights that can be uniquely generated from cancer-derived iPSCs, and their clinical applications. Last, challenges to expanding the use of iPSC modeling in blood cancers will be discussed.

CD47 expressed on cells functions as a "don't eat me" signal by binding SIRPa on macrophages and blocking phagocytosis. Cancer cells including AML evade phagocytosis by overexpressing CD47, and high CD47 expression in AML has been associated with inferior outcomes. Here we studied the pre-clinical activity of the 5F9 (magrolimab), an antibody that blocks the CD47, combined with BCL-2 inhibitor venetoclax with azacitidine (VEN/AZA) in AML both in vitro and in vivo. We first tested the combined efficacy of VEN/AZA and 5F9 in vitro in phagocytosis assay, utilizing monocytes isolated from seven healthy donors by CD16 positive selection and inducing macrophage differentiation with recombinant human M-CSF (rhM-CSF, 500ng/ml) for 1 week. Three paired isogenic AML cell lines MOLM-13, VEN-resistant (VEN-RE) MOLM-13, and p53-mutant MOLM-13 harboring R248W mutation, were pre-treated with DMSO or combination of 10nM VEN and 1uM AZA for 24hrs. Leukemia cells were labeled with Calcein and co-cultured with macrophages for 1h at a ratio of 2:1 (T:M), followed by flow cytometry read-out, quantifying Calcein+ macrophages that engulfed leukemia cells. The active phagocytosis was calculated as the percentage of Calcein+CD206+ cells from total CD206+ cells (Figure 1A, experimental schema). The 5F9/VEN-AZA combination significantly increased phagocytosis not only in parental but also in VEN-RE and p53-mutant MOLM-13 cells when compared with 5F9 or VEN/AZA alone treatments (Figure 1B). The treatment of VEN or AZA induced AnnexinV in MOLM-13 cells which may function as a pro-phagocytic signal and contribute to the enhanced phagocytosis (Figure 1C). Further proteomics studies to identify additional "eat me" signals following VEN/AZA treatment are ongoing and will be presented. Next, we examined the therapeutic efficacy of 5F9 alone or combined with VEN/AZA in the AML patient-derived xenografts (PDX) models. NSG mice were engrafted with VEN-RE AML PDX #344 (no canonical AML mutations detected by targeted sequencing) and #574 (FLT3-ITD, DNMT3A, NPM1-mutated). After confirmed bone marrow (BM) engraftment by flow cytometry, mice were randomized to receive vehicle, 5F9 (10mg/kg/d, ip. twice a week, 3 weeks), VEN (50mg/kg/g, po. qd, 14 days) / AZA (2.5mg/kg/d, ip, qd, 7 days), or their combination. Circulating leukemia burden was monitored by hCD45 flow cytometry in peripheral blood (PB) (Figure 1D), and mice survival was followed. 5F9 reduced leukemia burden, delayed leukemia progression and significantly extended mice' survival in both models, with the median survival of 276.5 days of 5F9 group compared to 197 days of vehicle in #344, and 81.5 vs 52 days in #574 (Figure 1E ). On the contrary, VEN/AZA therapy had no or minimal impact on tumor burden or survival. Notably, the combination of 5F9 and VEN/AZA resulted in elimination of circulating blasts and significant extension of survival in both models, median of 385 days in #344 and 124.5 days in #574. In summary, the VEN/AZA combined with 5F9 increased phagocytosis in isogenic MOLM-13 cells in vitro regardless of VEN resistance or p53 status and prolonged the survival in vivo in VEN/AZA refractory AML PDX models. The mechanisms of increased phagocytosis by combination treatment and the induction of "eat-me signals" by VEN/AZA is currently under investigation and will be presented. This triplet combination is currently being tested in a clinical trial (NCT04435691, Daver et al., ASH 2021) with high preliminary efficacy. Figure 1 Figure 1. Disclosures Chao: Gilead Sciences, Inc.: Current Employment; TigaTx: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties; Hepatx Inc: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Iconovir Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Stanford University Medical School: Membership on an entity's Board of Directors or advisory committees; Foresite capital: Consultancy; Bioverge: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Chimera Bioengineering: Current equity holder in publicly-traded company. Maute: Gilead Sciences: Ended employment in the past 24 months. Andreeff: Syndax: Consultancy; Karyopharm: Research Funding; ONO Pharmaceuticals: Research Funding; Senti-Bio: Consultancy; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Medicxi: Consultancy; Aptose: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Oxford Biomedica UK: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy; Amgen: Research Funding. Daver: Hanmi: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novimmune: Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; ImmunoGen: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Konopleva: Rafael Pharmaceuticals: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Ascentage: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; KisoJi: Research Funding; Stemline Therapeutics: Research Funding; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Cellectis: Other: grant support; Sanofi: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding.

Intro: Relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) is associated with poor outcomes, and remains an area of unmet clinical need. Emerging data indicate that a key macrophage immune checkpoint termed CD47 can be effectively targeted in r/r DLBCL. CD47 is a potent "don't eat me" signal and impairs tumor cell phagocytosis by engaging the inhibitory macrophage receptor, SIRPα. Importantly, early clinical studies demonstrate that blocking CD47-SIRPα interactions with an anti-CD47 antibody (magrolimab) yields promising activity in r/r DLBCL patients, particularly when combined with rituximab, which further potentiates lymphoma cell phagocytosis via its Fc domain. Moving forward, it will be crucial to identify combinatorial strategies that improve the efficacy of anti-CD47 therapy in r/r DLBCL. Tumor-associated macrophages (TAMs) contribute to an immunosuppressive environment in cancer through secretion of cytokines, metabolites, and expression of immune checkpoints. Sustained PI3K-γ signaling is critical to maintain immune suppressive TAM functions, and PI3K-γ inhibition polarizes TAMs into a pro-inflammatory state that culminates in potent anti-tumor T cell responses, sensitizing cancers to PD-1 blockade therapy. We hypothesized that activating TAMs through PI3K-δ/γ inhibition would also augment their inherent phagocytic capacity and synergize with phagocytosis-targeting treatments such as CD47 blockade. Thus, we investigated the impact of the dual PI3K-δ/γ inhibitor, duvelisib, on expression of key phagocytic receptors and pathways in human macrophages, as well as its effect on enhancing lymphoma cell phagocytosis with the anti-CD47 antibody, magrolimab. Results: RNA sequencing of duvelisib-treated human monocyte-derived macrophages revealed that PI3K-δ/γ inhibition induced broad transcriptional changes suggestive of an activated macrophage state (Fig 1A), including upregulation of interferon response genes (IRF9) and costimulatory molecules (CD86), while decreasing expression of immune suppressive cytokines (IL10) and inhibitory ligands (PDL1). Moreover, duvelisib upregulated transcription of canonical pro-phagocytic receptors (LRP1, FCGR2A,CD44), lysosomal degradation components (LYZ, CTSS) and phosphatidylserine-bridging genes (MFG-E8, GAS6), while inhibiting metabolic pathways, most notably mTOR, each of which has been associated with increased and more efficient phagocytosis. Given these findings, we evaluated the effect of dual PI3K-δ/γ inhibition on macrophage phagocytosis of DLBCL cells in the context of CD47 blockade in vitro. Across a broad panel of human DLBCL cell lines and multiple monocyte donors, pre-treatment of macrophages with duvelisib significantly increased lymphoma cell phagocytosis by magrolimab (Fig 1B-C). This effect reflected a direct increase in the phagocytic capacity of treated macrophages, as lymphoma cells were not exposed to duvelisib. In separate experiments, we found that duvelisib rarely induced full apoptosis of DLBCL cells, but did promote the expression of "eat me" signals, such as calreticulin and phosphatidylserine on many of the cell lines examined (Fig 1D). Collectively, these results suggest that PI3K-δ/γ inhibition enhances the inherent phagocytic capacity of macrophages, while also sensitizing lymphoma cells for engulfment. Similarly, potent effects were not observed with PI3K-δ- or PI3K-γ-specific inhibitors, suggesting that combined PI3K-δ/γ inhibition is critical to mediating these beneficial effects. Finally, to determine whether duvelisib impacted the efficacy of magrolimab in vivo, we treated human DLBCL xenografts and observed that while duvelisib had minimal impact on lymphoma growth, the combination of duvelisib and magrolimab induced complete tumor rejection in a high proportion of mice, leading to increased survival compared to mice treated with magrolimab alone (Fig 1E). Conclusion: Collectively, our findings suggest that targeting the PI3K-δ/γ axis favorably induces key macrophage phagocytic pathways, while simultaneously upregulating important "eat me" signals on lymphoma cells. Together, these alterations lower the phagocytic threshold of TAMs and dramatically increase the efficacy of anti-CD47 therapy in DLBCL xenograft models. This work paves the way for early-phase studies of duvelisib and magrolimab in r/r DLBCL and other lymphomas. Disclosures Godfrey: Verastem: Research Funding; Merck: Research Funding; Gilead: Research Funding. Coma:Verastem Oncology, Inc: Current Employment, Current equity holder in publicly-traded company. Maute:Gilead Sciences, Inc.: Current Employment. Chao:Gilead Sciences: Current Employment. Pachter:Verastem Therapeutics: Current Employment. Kline:Kite/Gilead: Speakers Bureau; Merck: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

PURPOSE To evaluate the safety, pharmacokinetics, and pharmacodynamics of Hu5F9-G4 (5F9), a humanized IgG4 antibody that targets CD47 to enable phagocytosis. PATIENTS AND METHODS Adult patients with solid tumors were treated in four cohorts: part A, to determine a priming dose; part B, to determine a weekly maintenance dose; part C, to study a loading dose in week 2; and a tumor biopsy cohort. RESULTS Sixty-two patients were treated: 11 in part A, 14 in B, 22 in C, and 15 in the biopsy cohort. Part A used doses that ranged from 0.1 to 3 mg/kg. On the basis of tolerability and receptor occupancy studies that showed 100% CD47 saturation on RBCs, 1 mg/kg was selected as the priming dose. In subsequent groups, patients were treated with maintenance doses that ranged from 3 to 45 mg/kg, and most toxicities were mild to moderate. These included transient anemia (57% of patients), hemagglutination on peripheral blood smear (36%), fatigue (64%), headaches (50%), fever (45%), chills (45%), hyperbilirubinemia (34%), lymphopenia (34%), infusion-related reactions (34%), and arthralgias (18%). No maximum tolerated dose was reached with maintenance doses up to 45 mg/kg. At doses of 10 mg/kg or more, the CD47 antigen sink was saturated by 5F9, and a 5F9 half-life of approximately 13 days was observed. Strong antibody staining of tumor tissue was observed in a patient at 30 mg/kg. Two patients with ovarian/fallopian tube cancers had partial remissions for 5.2 and 9.2 months. CONCLUSION 5F9 is well tolerated using a priming dose at 1 mg/kg on day 1 followed by maintenance doses of up to 45 mg/kg weekly.

BACKGROUND: The Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin’s lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically. METHODS: We conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin’s lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose. RESULTS: A total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing. CONCLUSIONS: The macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; ClinicalTrials.gov number, NCT02953509.)

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

Background Magrolimab (previously named 5F9) is a first-in-class antibody targeting CD47, a macrophage immune checkpoint and "don't eat me" signal on cancers. CD47 blockade induces tumor phagocytosis and eliminates leukemia stem cells (LSC) in AML models. Azacitidine (AZA) synergizes with magrolimab by inducing "eat me" signals on AML, to enhance phagocytosis. A Phase 1b trial of magrolimab+AZA was initiated in MDS/AML patients with preliminary reported results mainly from the safety cohort demonstrating high response rates in both diseases. Here we report data from the expansion cohort of this ongoing trial. Methods Results from this Phase 1b reported here focus on treatment of magrolimab+AZA in untreated intermediate to very high risk MDS patients by IPSS-R and untreated AML (induction chemotherapy ineligible) patients. A magrolimab priming/intrapatient dose escalation regimen (1-30 mg/kg weekly) was utilized to mitigate on target anemia. AZA dosing was 75mg/m2 days 1-7 on a 28 day cycle. Responses were assessed by IWG 2006 and ELN 2017 criteria for MDS and AML patients, respectively. Results 43 patients (18 MDS and 25 AML) with a median of 73 years of age were treated with magrolimab+AZA. 19% were intermediate cytogenetic risk with 63% poor risk (19% unknown). 28% of patients harbored a TP53 mutation. Magrolimab+AZA was well-tolerated with a safety profile similar to AZA monotherapy. Treatment-related AEs (>15% of patients) for magrolimab+AZA were anemia (37%), neutropenia (26%), and thrombocytopenia (26%). Treatment-related febrile neutropenia occurred in only 1 (2%) patient. Only 1 patient discontinued due to an AE. 29 patients were evaluable for efficacy at time of data cut. 13/13 (100%) untreated MDS patients had an objective response with 7 patients (54%) achieving a CR, 5 (39%) with marrow CR (3/5 also had hematologic improvement (HI)), and 1 (7%) with HI alone. In AML, 11/16 (69%) had an objective response; 8/16 (50%) with CR or CRi, 2 (13%) with PR, 1 (6%) with MLFS, and 5 (31%) with stable disease. Time to response was more rapid (median 1.9 mos) than expected for AZA alone. For those with abnormal cytogenetics at baseline, 40% and 44% of MDS and AML patients achieved a cytogenetic CR, respectively. 4/8 (50%) AML patients with CR/CRi and 2/12 (17%) MDS patients with CR or marrow CR were MRD negative by flow cytometry. 11/16 (69%) AML patients became RBC transfusion independent and 11/13 (85%) MDS patients had hematologic improvement. Given that CD47 is an LSC marker on leukemic cells, CD34+CD38- putative LSC frequency was measured by flow cytometry in the bone marrow in 5F9+AZA treated AML/MDS patients. In data available for analysis, LSCs were completely eliminated in 10/16 (63%) of AML/MDS patients who had a clinical response. Lastly, mutational analyses are ongoing to correlate subgroups with response. Interestingly, 7/8 (88%) evaluable TP53 mutant patients (5/6 AML patients [5 CR/CRi], 2/2 MDS [1 CR, 1 marrow CR]) achieved an objective response, highlighting efficacy in a poor prognosis and therapy-refractory population. No median duration response or overall survival has been reached for either MDS or AML patients with a median follow-up of 4.9 months (range 3.1 - 8.8 months) for MDS and 5.8 months (range 1.9 - 9.5 months) for AML. Conclusions Magrolimab+AZA is a novel immunotherapy regimen that blocks a key macrophage checkpoint. The combination therapy continues to be well tolerated with robust activity in MDS and AML patients with an ORR of 100% and 69%, respectively. High rates of putative LSC eradication suggest potential durable responses, with no median duration of response yet reached. Initial data indicate that 5F9+AZA may be particularly effective in TP53 mutant patients, a treatment-refractory subgroup. Expansion cohorts are ongoing (NCT03248479) with registrational studies in MDS being initiated. Additional patients, follow-up, and mutational characterization will be reported at time of presentation. Funded by Forty Seven and the California Institute for Regenerative Medicine. Disclosures Sallman: Celyad: Membership on an entity's Board of Directors or advisory committees. Lee:Bayer: Research Funding; Roche: Research Funding; Abbvie: Research Funding; Forty Seven, Inc.: Research Funding; Tolero: Research Funding. Daver:Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Agios: Consultancy; Pfizer: Consultancy, Research Funding; Servier: Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; NOHLA: Research Funding; Astellas: Consultancy; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Glycomimetics: Research Funding. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Komrokji:Novartis: Speakers Bureau; Incyte: Consultancy; JAZZ: Consultancy; Agios: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; JAZZ: Speakers Bureau. Van Elk:Forty Seven, Inc.: Employment, Equity Ownership. Lin:Forty Seven, Inc.: Employment, Equity Ownership. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Vyas:Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Forty Seven, Inc.: Research Funding; Daiichi Sankyo: Speakers Bureau; Abbvie: Speakers Bureau; Astellas: Speakers Bureau.

Introduction Magrolimab (Hu5F9-G4, 5F9) is a first-in-class IgG4 antibody targeting CD47, a macrophage immune checkpoint and "don't eat me" signal expressed on cancer cells. Blockade of CD47 leads to phagocytosis of tumor cells. Magrolimab synergizes with rituximab to eliminate CD20-positive lymphoma by enhancing antibody-dependent cellular phagocytosis. Magrolimab +rituximab demonstrated encouraging safety and efficacy in a Phase (Ph)1b dose escalation cohort in patients with relapsed/refractory (r/r) DLBCL and FL that were rituximab-refractory (Advani et al., NEJM 2018). CD24 is an additional "don't eat me" signal, and has been proposed as a potential target for immunotherapy (Barkal et al. Nature 2019). Here we describe immunohistochemical analysis of CD47 and CD24 in primary patient biopsies from an ongoing follow-up Phase 2 trial of Non-Hodgkin's lymphoma (including DLBCL and indolent lymphoma) patients treated with magrolimab+rituximab. Results By immunohistochemistry, 54 out of 54 patients assessed were positive for expression of CD47 at screening. High levels of CD47 expression were maintained during treatment with clinically-efficacious doses of magrolimab. Therapeutic response did not correlate with CD47 H-score expression levels. At screening, patients presented with highly variable levels of CD24 expression, with 40 of 54 samples showing positive staining in >30% of cells. We observed no significant correlation between CD24 expression by H-score and response to therapy (complete response + partial response) in either DLBCL or indolent lymphoma. Conclusions CD47 shows consistently high expression in primary biopsies from Non-Hodgkin's lymphoma patients and remains persistently high during treatment. In our Phase 2 trial, therapeutic response did not correlate with CD47 expression levels, suggesting that the degree of target expression is not a primary driver of magrolimab efficacy in Non-Hodgkin's lymphoma. Although we observe wide variation in CD24 expression within this cohort, its levels are not predictive of outcome for this disease indication. We are evaluating the expression of additional biomarkers to identify those Non-Hodgkin's lymphoma patients most likely to benefit from magrolimab+rituximab combination therapy. Disclosures Maute: Forty Seven Inc.: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Marjon:Forty Seven Inc.: Employment, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

Most lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell-derived lymphoma is repressed following activation of estrogen receptor β (ERβ, ESR2). By using ERβ-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERβ agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERβ reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERβ inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERβ activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERβ is expressed in primary MCL tissue. These results suggest that targeting ERβ with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

CD47 exerts different effects on disease in distinct cell types and locations and during different stages of experimental autoimmune encephalomyelitis., Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47−/− mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRPα and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL. Cancer Res; 71(4); 1374–84. ©2010 AACR.

CD47 is an anti-phagocytic (i.e. "don't eat me") signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential. We previously established that administration of 5F9 clears a subset of aged RBCs, resulting in a compensatory reticulocytosis. This occurs transiently, predominantly after the first dose, and does not worsen with subsequent doses. These findings, aligned with the observations of Oldenborg et al., which established that removal of aged RBCs, is dependent on the balance between the accumulating pro-phagocytic signals and loss of inhibitory anti-phagocytic signals. We therefore hypothesized that an initial lower 5F9 "priming dose", sufficient to trigger clearance of aged RBCs, would yield an overall younger pool of RBCs that are less vulnerable to subsequent higher 5F9 therapeutic "maintenance doses". Indeed, this priming and maintenance dosing strategy has substantially mitigated the on-target anemia and has allowed for 5F9 treatment in multiple clinical trials and indications. To further probe this observed selective RBC clearance, we employed a receptor occupancy assay to quantitate the CD47 expression on the cell surface of RBCs, leukocytes (WBCs), and acute myeloid leukemia (AML) blasts. We discovered that the priming dose of 5F9 not only triggered clearance of a subset of RBCs, but also resulted in a near complete loss of CD47 - a phenomenon we term CD47 pruning. This effect was RBC-specific, as WBCs and AML bone marrow (BM) blasts did not exhibit this same CD47 pruning (Fig 1). To explore the functional consequence of these unexpected clinical findings, we investigated whether we could recapitulate them in a preclinical mouse model. Similar to the clinical setting, anti-mouse CD47 blocking Ab treatments exhibited a similar pruning effect and transient anemia. Using this model, we asked whether, in the context of anti-CD47 treatment, these pruned RBCs were protected compared to unpruned RBCs. We transfused fluorescently labeled RBCs from CD47 Ab-untreated and CD47-treated mice into animals receiving continuous anti-CD47 treatment. We observed that CD47-pruned RBCs exhibited a significantly longer half-life compared to unpruned RBCs, suggesting that CD47 pruning is protective for RBCs. To distinguish whether this protection is cell-intrinsic or cell-extrinsic, we transfused fluorescently labeled RBCs into CD47 Ab-untreated and -treated mice and observed that CD47-pruned RBCs are rapidly cleared in untreated recipients, suggesting that this treatment-induced protection of RBCs is cell extrinsic and a host-based alteration. Through depletion of distinct immune subsets by chemical depletion, antibody depletion, splenectomy, and partial hepatectomies, we demonstrated that host immune effector cells (e.g. red-pulp macrophages, neutrophils, T cells etc.) were not necessary for this pruning phenomenon. Critically, we also found that this CD47 pruning and resulting tolerance was Fc-independent. In order to understand whether these findings were generalizable to other anti-human CD47 blocking agents, we conducted similar studies in double humanized huCD47 and huSIRPa micewith additional agents and observed this same CD47 pruning and tolerance phenomenon. Thereby suggesting this represents a class-effect of this emerging potential therapy class. Notably, these clinical and pre-clinical findings have been RBC-specific and have not precluded anti-tumor efficacy. The loss of CD47 on RBCs after the priming dose also suggests that the potential risk of CD47 Ab-mediated RBC agglutination following subsequent maintenance dosing is substantially reduced. Unlike prior RBC antigen modulation reports, to our knowledge, these findings represent a novel RBC antigen depletion phenomenon that is independent of the known RBC regulatory compartments, including the spleen, liver, major immune effectors cells, complement, and is critically Fc-independent. These findings provide fundamental insight into the mechanism underlying how anti-CD47 Abs are tolerated without impairing therapeutic efficacy. Disclosures Chen: Forty Seven Inc: Consultancy, Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Schrier:Forty Seven Inc.: Consultancy. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Elrod:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

Researchers have long studied malignant tumors using experimental techniques that assume that cancers are composed of identical cells; however, growing evidence indicates that cancers are composed of functionally heterogeneous cells maintained by a stem celllike population. Identification of tumor-initiating, or cancer stem cells, has been largely guided by principles established for normal stem cells. Cancer stem cells must be capable of initiating tumors when transplanted into immunodeficient mice, and the resulting tumors must replicate the heterogeneity of the original tumor. Demonstration of the ability to self-renew, a key feature of stem cells, is achieved through serial passage of tumors in xenohosts. Given their potential roles in tumor initiation, maintenance, and metastasis, the impact of cancer stem cells on the practice of medicine is likely to be profound.

CD47 is a widely expressed cell surface protein that functions as a regulator of phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin's lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409).

In this issue of Blood, Rumi et al and Rotunno et al demonstrate that essential thrombocythemia (ET) patients with calreticulin mutations exhibit lower leukocyte and hemoglobin values, higher platelet counts, and a lower thrombosis risk vs JAK2-mutated ET. Calreticulin-mutated ET appears to be a distinct entity with a more indolent course.

Candida albicans is an opportunistic pathogen that has adapted uniquely to life in mammalian hosts. One of the host factors recognized by this yeast is hemoglobin, which binds to a specific cell surface receptor. In addition to its regulating the expression of adhesion receptors on the yeast, we have found that hemoglobin induces the expression of a C. albicans heme oxygenase (CaHmx1p). Hemoglobin transcriptionally induces the CaHMX1 gene independent of the presence of inorganic iron in the medium. A Renilla luciferase reporter driven by the CaHMX1 promoter demonstrated rapid activation of transcription by hemoglobin and (cobalt protoporphyrin IX) globin but not by apoglobin or other proteins. In contrast, iron deficiency or exogenous hemin did not activate the reporter until after 3 h, suggesting that induction of the promoter by hemoglobin is mediated by receptor signaling rather than heme or iron flux into the cell. As observed following disruption of the Saccharomyces cerevisiae ortholog, HMX1, a CaHMX1 null mutant was unable to grow under iron restriction. This suggests a role for CaHmx1p in inorganic iron acquisition. CaHMX1 encodes a functional heme oxygenase. Exogenous heme or hemoglobin is exclusively metabolized to alpha-biliverdin. CaHMX1 is required for utilization of these exogenous substrates, indicating that C. albicans heme oxygenase confers a nutritional advantage for growth in mammalian hosts.

Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

Understanding the contribution of abnormal genetic and epigenetic programs to acute myeloid leukemia (AML) is necessary for the integrated design of genetic and epigenetic targeted therapies. While epigenetic therapies have been used to treat AML, so far efficacy has been limited. To this point, the reversibility of epigenetic modifications in AML is poorly understood, as are the relative contributions of the leukemic genetic and epigenetic programs to disease pathogenesis. To investigate these questions, we sought to reprogram primary AML leukemic blasts into induced pluripotent stem cells (iPSCs) and assess the effect of epigenetic reprogramming on leukemic behavior. We generated iPSCs from several AML patients with 11q23/MLL translocations using classic iPSC reprogramming factors (Sox-2, Klf4, Oct4, and Myc). Reprogrammed AML cells generated iPSCs as indicated by classic pluripotent features including embryonic stem cell morphology, expression of pluripotent markers, and formation of teratomas in vivo. In addition, AML iPSCs retained all the karyotypic and genetic abnormalities of the original AML patient. Despite presence of these mutations, AML iPSCs were able to differentiate into multiple cell types with normal function. Thus, human AML cells can be epigenetically reprogrammed into a pluripotent state despite the presence of leukemic driver mutations. Surprisingly, when AML iPSCs were differentiated in CD43+45+ hematopoietic cells, a leukemia phenotype re-emerged. Differentiated hematopoietic cells from AML iPSCs exhibited exclusively granulocytic-monocytic differentiation in colony forming assays, and serial replating potential in contrast to control iPSCs. When transplanted into immunodeficient mice, hematopoietic cells from AML iPSCs formed aggressive myeloid leukemias as evidenced by peripheral blood and bone marrow CD33+ myeloid engraftment, enlarged spleens, secondary transplant potential, and death from fulminant leukemia. These data indicate that epigenetic reprogramming alone was insufficient to eliminate leukemic behavior. To further investigate these epigenetic changes during reprogramming, we performed DNA methylation and gene expression analysis using 450K BeadChip arrays and RNAseq, respectively. Unsupervised hierarchical clustering demonstrated clustering of undifferentiated AML iPSCs with control iPSCs. In contrast, hematopoietic cells differentiated from AML iPSCs clustered exclusively with primary AML cells. Through gene ontology enrichment analysis, hematopoietic differentiated AML iPSCs were enriched for hypomethylation and gene activation of hematopoietic and leukemogenesis gene sets, including MLL gene targets, as compared to undifferentiated AML iPSCs. These data demonstrate that DNA methylation and gene expression profiles are reset upon epigenetic reprogramming of AML cells, but re-emerge upon hematopoietic differentiation, coinciding with re-emergence of the leukemic phenotype. Lastly, we demonstrated that our AML iPSC model can be used to physically separate and functionally profile genetic subclones within an AML patient. In one patient, we identified distinct AML iPSC genetic subclones (KRAS wildtype and G13D mutant) that could be prospectively separated and demonstrated differential growth properties and therapeutic susceptibilities. To determine the clinical relevance of these subclones, we sequenced this patient at relapse. Strikingly, the KRAS mutant clone, which was dominant at diagnosis, was absent at disease relapse implying relative chemotherapy resistance and competitive outgrowth of the KRAS wildtype subclone. Indeed, in our AML iPSC model, the KRAS wildtype clone demonstrated increased resistance to cytarabine in colony assays. We then investigated in a cohort of AML patients whether cytarabine sensitivity differed between KRAS wildtype and mutant patients. Strikingly, KRAS AML patients were more resistant to cytarabine in vitro compared to KRAS G13D patients. Thus, our AML iPSC model predicted clonal relapse in this patient and identified differential subclonal sensitivity to chemotherapy as a mechanism for relapse. In summary, pluripotent reprogramming of AML resets leukemic methylation/gene expression patterns, facilitates clonal targeting, and predicts subclonal relapse. Disclosures Chao: Forty Seven Inc.: Employment, Equity Ownership, Patents & Royalties. Majeti:Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47-signal-regulatory protein-α pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies.

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA . By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.

Under normal physiologic conditions, cellular homeostasis is partly regulated by balancing pro- and anti-phagocytic signals. CD47 is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin lymphoma, and bladder cancer, allowing cancer cells to evade phagocytosis by the innate immune system. Blockade of CD47 with a monoclonal antibody enables phagocytosis of cancer cells and leads to in vivo tumor elimination, but leaves most normal cells unaffected. In order for target cells to be phagocytosed upon blockade of an anti-phagocytic signal, we postulate that the cells must also display a potent pro-phagocytic signal. Here we identify calreticulin as a pro-phagocytic signal highly expressed on the surface of several human cancers including acute myeloid and lymphoblastic leukemias, chronic myeloid leukemia, non-Hodgkin lymphoma (NHL), bladder cancer, glioblastoma, and ovarian cancer, but minimally expressed on most normal cells. Increased CD47 expression correlated with high calreticulin levels on cancer cells, and was necessary for protection from calreticulin-mediated phagocytosis. Phagocytosis induced by anti-CD47 antibody required the interaction of target cell calreticulin with its receptor low density lipoprotein-receptor related protein (LRP) on phagocytic cells, as blockade of the calreticulin/LRP interaction prevented anti-CD47 antibody mediated phagocytosis. Lastly, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and NHL. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

SummaryMonoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44 + CK5 + CK20 − ). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% β-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

Many types of adult tissues, especially for high turnover tissues such as the blood and intestinal system, stand on a hierarchical tissue-specific stem cell system. Tissue-specific stem cells concurrently have self-renewal capacity and potential to give rise to all types of mature cells in their tissue. The differentiation process of the tissue-specific stem cell is successive restriction of these capacities. The first progeny of tissue-specific stem cells are multipotent progenitors (MPPs) that lose long-term self-renewal capacity yet have full lineage potential. MPPs in turn give rise to oligopotent progenitors, which then commit into lineage-restricted progenitors. This hierarchical system enables a lifelong supply of matured functional cells that generally have a short life span and a relatively high turnover rate. In this chapter, we review our findings and other key experiments that have led to the establishment of the current cellular stem and progenitor hierarchy in the blood-forming systems of mice and humans for both normal and leukemic hematopoiesis. We also review select signaling pathways intrinsic to normal hematopoietic and leukemic stem cell populations as well our recent findings elucidating the possible origin of the leukemia stem cell.

SummaryMacrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34 + CD38 − CD90 + Lin − ) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

Zhao et al[1][1] raise several issues from our prior work[2][2],[3][3] characterizing the mechanism of targeting CD47 in human hematopoietic malignancies. We reported that a blocking anti-CD47 antibody can eliminate multiple human tumor types and that a major mechanism is through phagocytosis via

Abstract 422 Background: Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells following activation, including NK cells. We hypothesized that the anti-lymphoma activity of rituximab could be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb. Methods: Rituximab induced upregulation of CD137 on NK cells was assessed using lymphoma cell lines and primary lymphoma patient samples. In-vitro NK cell degranulation and cytotoxicity were assessed by CD107a mobilization and chromium release. A murine lymphoma tumor model targeted by mouse anti-mouse CD20 mAb was used to assess in-vivo synergy of anti-CD20 and anti-CD137 mAbs. Mechanism of synergy was explored by T cell, NK cell, and macrophage depletion in the immune competent mouse model. A xenotransplant model in SCID mice with disseminated, luciferase-labeled lymphoma was used to demonstrate efficacy of anti-CD137 mAb and rituximab, and sufficiency of an innate immune response. Results: NK cells in human primary lymphoma samples do not express CD137 at baseline, however these cells highly upregulate CD137 when encountering rituximab-coated tumor B cells. Rituximab-induced NK cell degranulation and cytotoxicity as measured by CD107a mobilization (p=.006) and chromium release (p=.01) are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution (p Conclusions: Our results demonstrate the synergy of anti-CD137 mAb and rituximab by stimulation of rituximab-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against B cell malignancies by targeting first the tumor and then the host immune system. Disclosures: No relevant conflicts of interest to declare.

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative, and must be combined with cytotoxic chemotherapy for clinical benefit. Here we identify CD47 as an attractive monoclonal antibody target in NHL. A major function of CD47 is to inhibit phagocytosis, through binding its receptor, SIRP-alpha, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling by a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab through utilization of two independent antibody mechanisms will result in synergistic phagocytosis and elimination of NHL cells. We found that CD47 protein is highly expressed on primary human NHL cells compared to normal B cell counterparts. Higher CD47 gene expression predicted an inferior outcome in several cohorts of NHL patients, providing independent prognostic value. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro while the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells. In vivo, anti-CD47 antibody treatment of human NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control treatment. Furthermore, anti-CD47 antibody treatment in combination with rituximab led to elimination of lymphoma and cure of NHL-engrafted mice compared to either antibody treatment as a single agent. The above in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human lymphoma xenotransplants. These antibodies synergized through a novel mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2440.

Human acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self-renewing leukemia stem cells (LSC), which must be eliminated in order to cure the patient. We identified increased expression of CD47 on human AML LSC compared to their normal counterparts. CD47 is a cell surface molecule that serves as the ligand for SIRP-alpha on the surface of phagocytes, which in turn transmits a dominant inhibitory signal for phagocytosis. In this way, CD47 essentially functions as a “don't eat me” signal. We hypothesized that increased CD47 expression contributes to pathogenesis by inhibiting phagocytosis of AML LSC. Consistent with this hypothesis, we found that increased CD47 expression predicted worse overall survival in 3 independent cohorts of adult AML patients. Furthermore, we predicted that disruption of the interaction of CD47 with SIRP-alpha would result in phagocytosis and elimination of AML LSC. We found that blocking monoclonal antibodies directed against CD47 enabled phagocytosis of AML LSC, but not normal CD34+ human bone marrow progenitor cells, by human macrophages in vitro. Additionally, coating of human AML LSC with anti-CD47 monoclonal antibodies inhibited their engraftment in vivo in a xenotransplantation assay. Finally, analogous to a clinical therapy, treatment of human AML-engrafted mice with anti-CD47 antibody eliminated AML cells in the peripheral blood and bone marrow and targeted LSC. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with monoclonal antibodies capable of stimulating phagocytosis and elimination of LSC. Targeting of CD47 with blocking monoclonal antibodies to induce phagocytosis is a novel mechanism for antibody cancer therapy. While anti-CD47 antibodies can be effective monotherapy for human AML, such antibodies may be equally, if not more, effective as part of a combination strategy. The combination of an anti-CD47 antibody, able to block a strong inhibitory signal for phagocytosis, with a second antibody able to bind an LSC-specific molecule and engage Fc receptors on phagocytes, thereby delivering a strong positive signal for phagocytosis, may result in a synergistic stimulus for phagocytosis and specific elimination of AML LSC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-03.