Cedric Dos Santos

Treatment of Acute Myeloid Leukemia in Elderly Patients-A Therapeutic Dilemma.

Older adults represent the majority of approximately 20,000 new patients diagnosed with acute myeloid leukemia (AML) in the United States each year. While the treatment goal for younger patients is to achieve a cure with intensive therapeutic protocols, including standard chemotherapy and hematopoietic stem cell transplantation, these goals are less well defined in the elderly population. This is in part due to the continuous decline in treatment outcomes with increasing age secondary to a number of patient-related and disease-specific factors, ranging from the presence of comorbid conditions to the higher frequency of adverse cytogenetic and unfavorable molecular markers. Although best supportive care, low-dose cytarabine, and epigenetic drugs represent well recognized treatment concepts, no universally accepted strategy for the management of elderly patients with AML exists. Therapeutic decisions are widely based on the patients age, general health, the disease features, as well as the patients personal wishes. The predicament of treating AML in the elderly population is the central theme of this review.

Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option

Immune checkpoints are promising targets in cancer therapy. Recently, poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2) have been identified as novel immune checkpoints. In this investigation we show that acute myeloid leukemia (AML) cell lines and AML patient samples highly express the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ligands PVR and PVRL2. Using two independent patient cohorts, we could demonstrate that high PVR and PVRL2 expression correlates with poor outcome in AML. We show for the first time that antibody blockade of PVR or PVRL2 on AML cell lines or primary AML cells or TIGIT blockade on immune cells increases the anti-leukemic effects mediated by PBMCs or purified CD3+ cells in vitro. The cytolytic activity of the BiTE® antibody construct AMG 330 against leukemic cells could be further enhanced by blockade of the TIGIT-PVR/PVRL2 axis. This increased immune reactivity is paralleled by augmented secretion of Granzyme B by immune cells. Employing CRISPR/Cas9-mediated knockout of PVR and PVRL2 in MV4-11 cells, the cytotoxic effects of antibody blockade could be recapitulated in vitro. In NSG mice reconstituted with human T cells and transplanted with either MV4-11 PVR/PVRL2 knockout or wildtype cells, prolonged survival was observed for the knockout cells. This survival benefit could be further extended by treating the mice with AMG 330. Therefore, targeting the TIGIT-PVR/PVRL2 axis with blocking antibodies might represent a promising future therapeutic option in AML.

A genome-wide CRISPR screen identifies genes critical for resistance to FLT3 inhibitor AC220

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402-13. ©2017 AACR.

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.

Abstract 2470: Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

CD47 is a ubiquitously expressed cell surface Ig superfamily member. CD47 mediates a variety of biological processes, including leukocyte adhesion/migration, T-cell activation, apoptosis, and phagocytosis due to physical interaction with various proteins (integrins, thrombospondin-1, and signal regulatory protein alpha [SIRPα]). The CD47-SIRPα interaction negatively regulates phagocytosis. Enhanced CD47 expression has been described on hematological and solid cancers, suggesting that CD47 may mediate cancer cell escape from immune surveillance (e.g., by macrophages or neutrophils). Published studies demonstrated that anti-CD47 antibodies induced phagocytosis or apoptosis of cancer cells in vitro and mediated in vivo anti-tumor efficacy. Hypothesizing that blocking the CD47-SIRPα interaction will lead to phagocytosis and elimination of tumor cells, we generated 23 unique anti-CD47 monoclonal antibodies (mAbs), which potently block the CD47-SIRPα interaction. Upon further functional testing and optimization, three candidates emerged with nanomolar affinity to human and cynomolgus monkey CD47 and lack of hemagglutination and platelet aggregation activity: C47B157, C47B161 and C47B222 (human CD47 KD 3.53, 2.87, and 1.12 nM, respectively). To further characterize these antibodies and to better understand the contribution of merely blocking the CD47-SIRPα interaction to in vitro and in vivo anti-tumor activity, the mAbs were cloned into an effector function silent (IgG2sigma) and competent (IgG1) Fc backbone. In vitro ADCP assays demonstrated that IgG1 C47B157, C47B161, and C47B222 enhanced phagocytosis 4-fold over PBS control, while the IgG2sigma mAbs did not enhance phagocytosis. Subsequently, the anti-tumor activity was characterized in three human acute myeloid leukemia (AML) xenograft models (HL60/NSG, MV4-11/NSG, and Kasumi-3/NSG mice). At 10mg/kg C47B157, C47B161, and C47B222 completely suppressed growth of leukemia cells across all models in peripheral blood as IgG1 (0.74-2.78%) vs. control (23.2-88.5%) at time of control sacrifice. As IgG2sigma, C47B222 caused greatest suppression at 10 mg/kg in all models (1.6-2.2%). Additional in vivo studies with either AML cell lines or AML patient-derived primary cells revealed that although IgG2sigma C47B222 reduced disease outgrowth in peripheral blood and distinct organs (e.g., spleen), merely blocking the CD47-SIRPα interaction did not yield a survival advantage. In non-human primates, a single dose of IgG1 C47B222 caused ∼40% decrease in red blood cell count, hematocrit and hemoglobin at 1 mg/kg. In contrast, IgG2sigma C47B222 had minimal impact on red cell indices at 1 and 10 mg/kg. In conclusion, the findings suggest that efficacy and tolerability of anti-CD47 mAbs are Fc effector function dependent. The need to balance efficacy and tolerability raise questions on the feasibility of targeting CD47 in the development of cancer therapeutics. Citation Format: E. Christine Pietsch, Jianying Dong, Xiaochun Zhang, Diana Chin, Rebecca Hawkins, Thai Dinh, Mimi Zhou, Brandy Strake, Ping-Hua Feng, Meredith Rocca, Cedric Dos Santos, Xiaochuan Shan, Gwenn Danet-Desnoyers, Elizabeth Kaiser, Hillary Quinn, Jeffrey A. Nemeth, Ricardo Attar. Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2015-2470