slan+ Monocytes Kill Cancer Cells Coated in Therapeutic Antibody by Trogoptosis.

Monocytes positive for 6-Sulfo LacNAc (slan) are a major subset of nonclassical CD14dimCD16+ monocytes in humans. We have shown that slan+ cells infiltrate lymphomas and elicit an antibody-dependent cellular cytotoxicity (ADCC) of neoplastic B cells mediated by the anti-CD20 therapeutic rituximab. Herein, by performing blocking experiments and flow cytometry analyses, as well as confocal microscopy and live-cell imaging assays, we extended the findings to other humanized antibodies and deciphered the underlying effector mechanism(s). Specifically, we show that, after coculture with target cells coated with anti-CD20 or anti-CD38, slan+ monocytes mediate trogocytosis, a cell-cell contact dependent, antibody-mediated process that triggers an active, mechanic disruption of target cell membranes. Trogocytosis by slan+ monocytes leads to a necrotic type of target cell death known as trogoptosis, which, once initiated, was partially sustained by endogenous TNFα. We also found that slan+ monocytes, unlike natural killer (NK) cells, mediate a direct ADCC with all types of anti-CD47 analyzed, and this was independent of their IgG isotype. The latter findings unveil a potentially relevant contribution by slan+ monocytes in mediating the therapeutic efficacy of anti-CD47 in clinical practice, which could be particularly important when NK cells are exhausted or deficient in number. Overall, our observations shed new light on the cytotoxic mechanisms exerted by slan+ monocytes in antibody-dependent tumor cell targeting and advance our knowledge on how to expand our therapeutic arsenal for cancer therapy.

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia.

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, p. 252. This article is highlighted in the In This Issue feature, p. 247.

First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers.

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.

Induced pluripotent stem cell modeling of malignant hematopoiesis.

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.

Therapeutic Targeting of the Macrophage Immune Checkpoint CD47 in Myeloid Malignancies.

In recent years, immunotherapies have been clinically investigated in AML and other myeloid malignancies. While most of these are focused on stimulating the adaptive immune system (including T cell checkpoint inhibitors), several key approaches targeting the innate immune system have been identified. Macrophages are a key cell type in the innate immune response with CD47 being identified as a dominant macrophage checkpoint. CD47 is a "do not eat me" signal, overexpressed in myeloid malignancies that leads to tumor evasion of phagocytosis by macrophages. Blockade of CD47 leads to engulfment of leukemic cells and therapeutic elimination. Pre-clinical data has demonstrated robust anti-cancer activity in multiple hematologic malignancies including AML and myelodysplastic syndrome (MDS). In addition, clinical studies have been underway with CD47 targeting agents in both AML and MDS as monotherapy and in combination. This review will describe the role of CD47 in myeloid malignancies and pre-clinical data supporting CD47 targeting. In addition, initial clinical data of CD47 targeting in AML/MDS will be reviewed, and including the first-in-class anti-CD47 antibody magrolimab.

CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma.

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 .).

Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease.

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.

Myeloid Cell Origins, Differentiation, and Clinical Implications.

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.

A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

Two faces of ET: CALR and JAK2.

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.

Inhibition of lymphoma vascularization and dissemination by estrogen receptor ß agonists.

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 ß (ERß, ESR2). By using ERß-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 ERß 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 ERß reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERß inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERß activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERß is expressed in primary MCL tissue. These results suggest that targeting ERß with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

Treatment challenges in the management of relapsed or refractory non-Hodgkin's lymphoma - novel and emerging therapies.

Over the last few decades, advances in immunochemotherapy have led to dramatic improvement in the prognosis of non-Hodgkin's lymphoma (NHL). Despite these advances, relapsed and refractory disease represents a major treatment challenge. For both aggressive and indolent subtypes of NHL, there is no standard of care for salvage regimens, with prognosis after relapse remaining relatively poor. Nevertheless, there are multiple emerging classes of targeted therapies for relapsed/refractory disease, including monoclonal antibodies, antibody- drug conjugates, radioimmunotherapy, small-molecule inhibitors of cell-growth pathways, and novel chemotherapy agents. This review will discuss treatment challenges of NHL, current available salvage regimens for relapsed/refractory NHL, and the safety and efficacy of novel emerging therapies.

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

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.

The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications.

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.

Programmed cell removal: a new obstacle in the road to developing cancer.

The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy.

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.

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker.

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.

CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies.

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.