Therapy For Hematologic Malignancies Research Articles

CAR T-Cell Therapy in Hematological Malignancies.

Chimeric antigen receptor (CAR) T-cells (CAR T-cells) are a promising therapeutic approach in treating hematological malignancies. CAR T-cells represent engineered autologous T-cells, expressing a synthetic CAR, targeting tumor-associated antigens (TAAs) independent of major histocompatibility complex (MHC) presentation. The most common target is CD19 on B-cells, predominantly used for the treatment of lymphoma and acute lymphocytic leukemia (ALL), leading to approval of five different CAR T-cell therapies for clinical application. Despite encouraging clinical results, treatment of other hematological malignancies such as acute myeloid leukemia (AML) remains difficult. In this review, we focus especially on CAR T-cell application in different hematological malignancies as well as strategies for overcoming CAR T-cell dysfunction and increasing their efficacy.

Engineering strategies for broad application of TCR-T- and CAR-T-cell therapies.

Adoptive-cell therapy, including the transfer of tumor-infiltrating T lymphocytes after in vitro expansion or T cells redirected to tumor antigens using antigen-specific transgenic T-cell receptor T cells (TCR-T cells) or chimeric antigen receptor T cells (CAR-T cells), has shown a significant clinical impact. Particularly, several types of CAR-T-cell therapies have been approved for the treatment of hematological malignancies. The striking success of CAR-T-cell therapies in hematological malignancies motivates their further expansion to a wide range of solid tumors, yet multiple obstacles, including the lack of proper target antigens exhibiting a tumor-specific expression pattern and the immunosuppressive tumor microenvironment (TME) impairing the effector functions of adoptively transferred T cells, have prevented clinical application. Gene engineering technologies such as the CRISPR/Cas9 system have enabled flexible reprogramming of TCR/CAR-T-cell signaling or loading genes that are targets of the tumor immunosuppression as a payload to overcome the difficulties. Here, we discuss recent advances in TCR/CAR-T-cell engineering: various promising approaches to enhance the anti-tumor activity of adoptively transferred T cells in the TME for maximizing the efficacy and the safety of adoptive-cell therapy are now being tested in the clinic, especially targeting solid tumors.

FcεRIγ-negative NK cells persist in vivo and enhance efficacy of therapeutic monoclonal antibodies in multiple myeloma

AbstractMonoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (∼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.

Clinicopathological features and prognostic significance of programmed death ligand 1 in pediatric ALK-positive anaplastic large cell lymphoma: results of the ALCL99 treatment in Japan

Programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) blockade is a promising therapy for hematological malignancies. However, the association of PD-L1 expression with the clinicopathological features and prognosis in pediatric ALK-positive anaplastic large cell lymphoma (ALCL) remains unclear. Using PD-L1/ALK immunofluorescence double staining, we evaluated the PD-L1 expression on tumor cells/tumor-infiltrating immune cells (TIICs) and the quantity of TIICs in 54 children with ALK-positive ALCL treated with the ALCL99 protocol. The percentages of PD-L1-positive tumor cells were significantly lower in patients with skin/mediastinum involvement, clinical high-risk group, present minimal disseminated disease (MDD), and a low ALK-antibody titer. The percentages of PD-L1-positive TIICs were significantly higher in patients with absent MDD. The percentages of TIICs were significantly lower in patients with absent MDD and a common morphological pattern. We classified patients according to the PD-L1 expression on tumor cells (Tumor-PD-L1), PD-L1 expression on TIICs (TIIC-PD-L1), and quantity of TIICs (TIIC-quantity). The progression-free survival (PFS) did not differ between Tumor-PD-L1high and Tumor-PD-L1low ALCL; TIIC-PD-L1high and TIIC-PD-L1low ALCL; and TIIC-quantityhigh and TIIC-quantitylow ALCL. According to the combined parameters of Tumor-PD-L1 and TIIC-quantity, Tumor-PD-L1high/TIIC-quantityhigh ALCL had a worse 5-year PFS than other ALCL (50% versus 83%; P=.009). Tumor-PD-L1high/TIIC-quantityhigh ALCL remained a significant prognostic factor in multivariate analysis (P=.044). This is the first study to demonstrate that a high tumoral PD-L1 expression with a high quantity of TIICs was associated with a poor prognosis in pediatric ALK-positive ALCL. The tumor microenvironment of ALK-positive ALCL may be relevant to the clinicopathological features and prognosis.

Scalable Manufacturing of CAR T cells for Cancer Immunotherapy.

As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development.This review provides a critical appraisal of these technologies that can be leveraged to scale and optimize the production of next generation CAR T cells.

Identification of HSC/MPP expansion units in fetal liver by single-cell spatiotemporal transcriptomics.

Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP 'pocket-like' units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage-HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell-cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

Balancing the CAR T: perspectives on efficacy and safety of CAR T-cell therapy in hematologic malignancies.

Chimeric antigen receptor (CAR) T-cell therapy is an example of how immunotherapy is revolutionizing the treatment of hematologic malignancies with unprecedented response rates in patients with relapsed/refractory lymphoma, multiple myeloma, and acute lymphoblastic leukemia. The process for administering CAR T-cell therapy is complex, with multiple steps including CAR T-cell manufacturing, lymphodepleting chemotherapy, cellular therapy infusion, and management of short-term and long-term toxicities. While effective, broad use of CAR T-cell therapies is limited by potential for life-threatening toxicities, challenges related to manufacturing a patient-specific product, high costs and inadequate reimbursement, and incomplete or unsustained disease response. Pharmacists are intricately involved in the process of providing CAR T-cell therapy both at the organizational level of budgeting and coordinating therapy and in direct patient care roles providing counseling and support for adverse effect management. Research in CAR T-cell therapy is expected to improve tolerability and expand indications to more types of malignancies and earlier phases of disease. Managed care professionals should have an understanding of the clinical trial data and place in therapy in lymphoma, myeloma, and acute lymphoblastic leukemia as well as guideline recommendations for adverse effect management associated with CAR T-cell therapies.

Research Advances in the Mechanisms of BRD4 and Its Inhibitors in Hematologic Malignancies--Review

Bromodomain-containing protein 4 (BRD4) is one of the most important members in the bromodomain and extra terminal domain(BET) family, it plays an important role in cellular physiology in human body, such as cell cycles, cell proliferation, and immune response. Recent studies have shown that BRD4 is associated with occurrence and development of acute myeloblastic leukemia, multiple myeloma and lymphoma. The mechanisms of BRD4 in hematologic malignancies including the regulation of c-Myc expression, and participation of the composition of super-enhancer, etc. At present, many kinds of inhibitors have been developed to target inhibit BRD4 for therapy in hematologic malignancies, and some of BRD4 inhibitors have entered phase Ⅱ clinical trials, which suggested that BRD4 inhibitors are expected to become new therapeutic agents for hematologic malignancies. In this review, the research advance of BRD4 and BRD4 inhibitors in hematologic malignancies was summarized briefly.

Strengthening the CAR-T cell therapeutic application using CRISPR/Cas9 technology.

Adoptive cell immunotherapy with chimeric antigen receptor T (CAR-T) cell has brought a revolutionary means of treatment for aggressive diseases such as hematologic malignancies and solid tumors. Over the last decade, the United States Food and Drug Administration (FDA) approved five types of CAR-T cell therapies for hematologic malignancies, including Idecabtagene vicleucel (Abecma), Lisocabtagene maraleucel (Breyanzi), Brexucabtagene autoleucel (Tecartus), Tisagenlecleucel (Kymriah), and Axicabtagene ciloleucel (Yescarta). Despite outstanding results gained from different clinical trials, CAR-T cell therapy is not free from side effects and toxicities, and needs careful investigations and improvements. Gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, has emerged as a promising tool to address some of the CAR-T therapy hurdles. Using CRISPR/Cas9 technology, CAR expression as well as other cellular pathways can be modified in various ways to enhance CAR-T cells antitumor function and persistence in immunosuppressive tumor microenvironment. CRISPR/Cas9 technology can also be used to decrease CAR-T cell toxicities and side effects. Hereby, we discussed the practical challenges and hurdles related to the accuracy, efficiency, efficacy, safety, and delivery of CRISPR/Cas9 technology to the genetically engineered-T cells. Combining of these two state-of-the-art technologies, CRISPR/Cas9 and CAR-T cells, the field of oncology has an extraordinary opportunity to enter a new era of immunotherapy, which offers novel therapeutic options for different types of tumors.

Alternative splicing of BCL-X and implications for treating hematological malignancies.

BCL-X is a member of the BCL-2 family. It regulates apoptosis and plays a critical role in hematological malignancies. It is well-known that >90% of human genes undergo alternative splicing. A total of 10 distinct splicing transcripts of the BCL-X gene have been identified, including transcript variants 1–9 and ABALON. Different transcripts from the same gene have different functions. The present review discusses the progress in understanding the different alternative splicing transcripts of BCL-X, including their characteristics, functions and expression patterns. The potential use of BCL-X in targeted therapies for hematological malignancies is also discussed.

Cytokine Release Syndrome Associated with T-Cell-Based Therapies for Hematological Malignancies: Pathophysiology, Clinical Presentation, and Treatment.

Cytokines are a broad group of small regulatory proteins with many biological functions involved in regulating the hematopoietic and immune systems. However, in pathological conditions, hyperactivation of the cytokine network constitutes the fundamental event in cytokine release syndrome (CRS). During the last few decades, the development of therapeutic monoclonal antibodies and T-cell therapies has rapidly evolved, and CRS can be a serious adverse event related to these treatments. CRS is a set of toxic adverse events that can be observed during infection or following the administration of antibodies for therapeutic purposes and, more recently, during T-cell-engaging therapies. CRS is triggered by on-target effects induced by binding of chimeric antigen receptor (CAR) T cells or bispecific antibody to its antigen and by subsequent activation of bystander immune and non-immune cells. CRS is associated with high circulating concentrations of several pro-inflammatory cytokines, including interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors. Recently, considerable developments have been achieved with regard to preventing and controlling CRS, but it remains an unmet clinical need. This review comprehensively summarizes the pathophysiology, clinical presentation, and treatment of CRS caused by T-cell-engaging therapies utilized in the treatment of hematological malignancies.

Patient-Reported Symptom and Functioning Status during the First 12 Months after Chimeric Antigen Receptor T Cell Therapy for Hematologic Malignancies

Chimeric antigen receptor (CAR) T cell therapy is being increasingly used to treat patients with advanced hematologic malignancies; however, the symptoms related to standard of care CAR T cell therapy during the first year after treatment have not been assessed using patient-reported outcome (PRO) measurements. This study aimed to quantify patients' perspectives of symptom burden and functional status using PROs during the first year after CAR T cell therapy for hematologic malignancies, especially in patients who experienced grade 2-4 toxicities. Sixty patients were enrolled in this observational cross-sectional study at any time during their first 12 months post-treatment. All 60 had received CAR T cell therapy as standard of care at MD Anderson Cancer Center in 2019. PROs were measured using the MD Anderson Symptom Inventory (MDASI), the PROs Measurement Information System 29 (PROMIS-29), the global health tool EQ5D-5L, and the single-item health-related quality of life scale (HRQoL). Twenty-two additional symptoms related to CAR T cell therapy, as identified by an expert panel, were also evaluated. CAR T cell therapy-related toxicities were rated according to the ASTCT consensus grading criteria. The majority of patients (52 of 60; 87%) received axicabtagene ciloleucel (Yescarta). One-third of the patients developed grade 2-4 cytokine release syndrome or neurotoxicity. The first 90 days after infusion represented the most symptomatic period, in which >10% of patients rated 18 symptoms as severe (ie, MDASI symptom score of 7 to 10 on scale of 0 to 10), strongly indicating the need for effective symptom management. Physical functioning, measured by interference on the "general activity" item on the MDASI and this domain on the PROMIS-29, were significantly worse in patients who underwent therapy during the first 30 days compared with those who underwent therapy over 90 days (all P < .05 with the Hochberg step-up procedure), whereas the EQ5D-5L and single-item HRQoL did not detect such differences. Compared with patients who had mild cytokine release syndrome or neurotoxicity (grade 0-1), patients who developed grade 2-4 toxicities persistently reported multiple severe symptoms after 30 days following therapy (all P < .05). Furthermore, although using a different recall period, patient-reported scores on several PROMIS-29 domains were significantly correlated with the scores of corresponding MDASI symptom items. This real-world quantitative PRO symptoms study provides evidence of unique profiles of the physical, psychological, and cognitive symptom burden in patients undergoing CAR T cell therapy that varies within the first year after infusion and demonstrates differences among PRO measurement scales. These results support the need for validation of fit-for-purpose PRO measurements for routinely monitoring symptom and toxicity burdens in CAR T cell therapy care settings.

The Effects of Interferons on Allogeneic T Cell Response in GVHD: The Multifaced Biology and Epigenetic Regulations.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. This beneficial effect is derived mainly from graft-versus-leukemia (GVL) effects mediated by alloreactive T cells. However, these alloreactive T cells can also induce graft-versus-host disease (GVHD), a life-threatening complication after allo-HSCT. Significant progress has been made in the dissociation of GVL effects from GVHD by modulating alloreactive T cell immunity. However, many factors may influence alloreactive T cell responses in the host undergoing allo-HSCT, including the interaction of alloreactive T cells with both donor and recipient hematopoietic cells and host non-hematopoietic tissues, cytokines, chemokines and inflammatory mediators. Interferons (IFNs), including type I IFNs and IFN-γ, primarily produced by monocytes, dendritic cells and T cells, play essential roles in regulating alloreactive T cell differentiation and function. Many studies have shown pleiotropic effects of IFNs on allogeneic T cell responses during GVH reaction. Epigenetic mechanisms, such as DNA methylation and histone modifications, are important to regulate IFNs’ production and function during GVHD. In this review, we discuss recent findings from preclinical models and clinical studies that characterize T cell responses regulated by IFNs and epigenetic mechanisms, and further discuss pharmacological approaches that modulate epigenetic effects in the setting of allo-HSCT.

Abstract 63: Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, translating these results in solid tumors remains challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Herein, we established a fully immunocompetent syngeneic mouse model and evaluated the interaction of CAR-M with the tumor microenvironment (TME) and the endogenous adaptive immune system. Murine bone marrow-derived CAR-expressing macrophages (muCAR-M) were efficiently engineered to express an anti-huHER2 CAR using the chimeric adenoviral vector Ad5f35. MuCAR-M, but not control untransduced (UTD) macrophages, specifically phagocytosed HER2+ target cancer cell lines and killed HER2-expressing murine CT26 colorectal and human AU-565 (HER2+) breast cancer cells in a dose-dependent manner. Moreover, CAR-M induced MHC-I and MHC-II expression on tumor cells and cross-presented tumor-associated antigens (TAA) resulting in CD8 T cell activation. To evaluate muCAR-M in an immunocompetent in vivo setting, BALB/c mice were engrafted with subcutaneous CT26-HER2+ tumors and treated with HER2-CAR or UTD macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cells reactive to the CT26 MHC-I antigen gp70, indicating enhanced epitope spreading. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2- CT26 rechallenge model, indicating the induction of long-term T cell memory against TAA. To evaluate the systemic anti-tumor immune response, we simultaneously engrafted BALB/c mice with CT26-HER2+ and CT26-Wt tumors on opposite flanks and treated mice with local administration of CAR-M restricted to the HER2+ tumors. After CAR-M treatment, 75% of mice cleared their CT26-HER2+ tumors and the growth rate of the contralateral CT26-Wt tumors was significantly reduced, demonstrating an abscopal effect. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with PD1 checkpoint inhibitor therapy in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further improved tumor control and overall survival. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Linara Gabitova, Yumi Ohtani, Olga Shestova, Saar Gill, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 63.

Abstract 62: Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC)

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. Treatment of RCC has improved dramatically over the last decade, however, a curative treatment for advanced RCC remains rare. Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug”. The FDA approval of CAR-T cell therapies have ushered this new type of cellular immunotherapy into mainstream cancer therapy for hematologic malignancies. To date, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the immunosuppressive tumor microenvironment (TME), and on-target off-tumor toxicities due to shared antigens on normal tissues. Carbonic anhydrase IX (CAIX) is a therapeutic target against ccRCC that is under the control of HIF1α and becomes overexpressed on the surface of ccRCC cells because of VHL inactivation. The first anti-CAIX CAR-T study using the 1st generation G250-CD3 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC was terminated due to hepatitis that developed after CAR-T cell infusions. The on-target off-tumor side effects were attributed to low expression of CAIX on healthy bile duct cells. To translate CAR-T cell therapy to ccRCC, we designed dual-targeted fine-tuned immune restoring (DFIR) CAR-T cells. The DFIR-CAR T cells secreted immune checkpoint inhibitor (ICI) monoclonal antibodies at the tumor site and exhibited superior efficacy and safety profiling. Increased efficacy is achieved through anti-CD70/CAIX dual-targeting CAR which allows the CAR-T cell activation in response to either antigen to mitigate solid tumor heterogeneity. Elevated safety is addressed through fine-tuned CARs which have the affinities of the scFv targeting moieties tailored so that they are activated only by high density tumor associated antigens (TAAs) but not the same antigens expressed at physiologic levels on normal tissues. The ICI payloads act globally on the TME, not only to prevent CAR-T cell exhaustion but also to restore anti-tumor activity of the educated tumor infiltrated lymphocytes (TILs) that have accumulated in the TME. In summary, our DFIR CAR-T cell therapy holds the promise to achieve cures of ccRCC by killing heterogenous ccRCC cells, mitigating against on-target off-tumor toxicity, reversing TME immunosuppression and restoring host anti-tumor immunity. We believe that DFIR-CAR T cells are ready to be translated to the clinic upon completion of pre-IND studies. Citation Format: Yufei Wang, Alicia Buck, Marion Grimaud, Sreekumar Kodangattil, Cecile Razimbaud, Atef Fayed, Matthew Chang, Aedin Culhane, David A. Braun, Toni K. Choueiri, Catherine J. Wu, Kevin S. Wei, Leo L. Chan, Brandon P. Piel, Elena V. Ivanova, Cloud P. Paweletz, David A. Barbie, Rebecca Jennings, Miriam Ficial, Maura Aliezah Sticco-Ivins, Sabina Signoretti, Gordon J. Freeman, Quan K. Zhu, Wayne A. Marasco. Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 62.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Phase 1 Clinical Trial Methodology in Hematological Malignancies from 2005 - 2015: A Systematic Review

Background:Current literature suggests that innovative model-based dose escalation schemes in phase 1 trials estimate the maximum tolerated dose of a drug more precisely and efficiently. Additional strategies to meet enrollment needs for phase I studies include multi-center trial design, broader inclusion of patients, and use of expansion cohorts.There has been a significant growth in novel therapies in hematological malignancies over recent years. However, it is unclear whether newer phase I trial methodologies have been employed to assess these treatments.Methods:A systematic review of MEDLINE and EMBASE between 2005 and 2015 was performed to identify all phase I and phase I/II trials in adult patients with hematologic malignancies. Trials involving autologous and allogeneic transplants were excluded. Data extracted for analysis included number of trial centers (single vs. multi), use of an innovative dose-escalation design, use of expansion cohorts (i.e. phase Ib, phase II), and inclusion of patients over age 80. Innovative dose-escalation design was defined as designs that used a model based approach or a more sophisticated rule based design other than traditional 3+3 Phase I designs. Studies published from 2005-2010 were compared with studies from 2011-2015. The chi-squared test was used to compare the analyzed binary variables.Results:We identified 568 phase 1 clinical trials in malignant hematology meeting specified criteria which had published methodologies available for review. Results are summarized in Table 1. There were more phase I trials published in 2011-2015 (347) compared with 2005-2010 (222). From 2011-2015, compared to the 2005-2010 period, there was a significant increase in the proportion of trials which were multicenter (66.2% vs 54.1%, p=0.003), trials utilizing innovative dose escalation designs (9.5% vs 5%, p=0.046), and trials utilizing expansion cohorts (47.8% vs 35.1%, p=0.0025). There was a non-significant trend toward increased number of trials which included patients over age 80 (51.3% vs. 42.7%, p=0.055).Conclusions:Among phase I trials in malignant hematology over 2005-2015, there has been an increase in use of novel methodologies including multicenter collaboration, innovative dose escalation designs, and use of expansion cohorts, although many of these features remain underutilized.The number of studies has increased at a rapid rate, likely reflecting recent development of molecularly targeted agents and immunotherapy. Although it is possible that the prevalence of many hematologic malignancies is increasing, it is unlikely that it matches the rate of growth of phase 1 trials, thus making it important to utilize more efficient trial designs in order to meet enrollment goals. [Display omitted] DisclosuresYee:Novartis Canada: Honoraria; Astex: Research Funding; Celgene Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Oncoethix: Research Funding. Chavez:Janssen: Speakers Bureau; Abbvie: Speakers Bureau; Kite: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees.

Tocilizumab Is Safe after T-Cell Replete Peripheral Blood Haploidentical Donor Transplantation

Introduction: Allogeneic hematopoietic cell transplantation is a cornerstone of therapy for hematologic malignancies and often a patient's only curative intent treatment. Overall outcomes for haploidentical transplantation appear to be excellent, however, this novel approach brings toxicities that are particular to its biological and clinical milieu. We previously described occurrence of severe cytokine release syndrome (CRS) after haplo-HCT. Severe CRS was associated with poor clinical outcomes, including transplant related mortality (TRM), overall survival (OS), and neutrophil engraftment (Abboud et al, BBMT, 10/2016). Furthermore, tocilizumab rapidly reversed the signs and symptoms of the dysregulated inflammatory reaction in most cases of CRS.Here we compare short and long term complications in peripheral blood haplo-HCT patients who did and did not receive tocilizumab.Patients and Methods: We performed a retrospective review of patients who underwent haplo-HCT transplantation at our institution from July 2009 through January 2017. A total of 169 patients were identified, 9% (15) received tocilizumab for treatment of CRS. We compared rates of acute and chronic GVHD, time to relapse, and incidence infection related deaths between patients who received tocilizumab and those who did not.Results: We found no difference in acute GVHD (hazard 1.08, p 0.83), time to relapse (hazard 1.06, p 0.90), or incidence of infection related deaths (hazard 0.764, p 0.65) between patients who received tocilizumab and those who did not.There was a trend towards a lower rate of chronic GVHD in patients who received tocilizumab compared with those who did not, with a hazard of 0.56 (95% CI 0.158 - 0.99, p= 0.37).Conclusions: Tocilizumab is safe following haplo-SCT. Tocilizumab did not increase the risk of acute GVHD, chronic GVHD, time to relapse, or incidence of infection related deaths. There was a trend towards a lower rate of chronic GVHD in patients who received tocilizumab. [Display omitted] DisclosuresUy:Boehringer Ingelheim: Consultancy; Novartis: Consultancy, Other: Travel Suppport; GlycoMimetics: Consultancy. Vij:Celgene: Honoraria; Konypharma: Honoraria; Bristol-Meyers-Squibb: Honoraria; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Janssen: Honoraria.

Engineering of Induced Pluripotent Stem Cell-Derived Hemogenic Endothelium

Background: Autologous cellular therapy of hematological malignancies and primary immunodeficiencies has been limited by the inability to produce functional hematopoietic stem cells (HSCs) from patient-derived induced pluripotent stem cells (iPS). Previous approaches have failed to demonstrate multi-lineage potential or secondary bone marrow engraftment in murine hosts - the gold standards for demonstrating HSC self-renewal and differentiation. Building upon recent evidence that HSCs derive from the definitive hemogenic endothelium (HE) of the embryonic lateral plate mesoderm, we set out to engineer iPS-derived HSCs by directed differentiation, and to investigate potential applications for modeling genetic blood diseases in vitro .Methods: Following embryoid body formation, human iPS derived from bone marrow mesenchymal stem cells underwent stepwise, morphogen-directed differentiation into HE, followed by polycistronic lentiviral transduction with five transcription factors (RUNX1, ERG, LCOR, HOXA5, HOXA9) on day 3 of endothelial-to-hematopoietic transition (EHT) induction, to enable further conversion to HSC-like cells with lymphoid potential in vitro and known multi-lineage engraftment capacity in vivo . A candidate screen of 17 cytokines, epigenetic modifiers and small molecules aimed at inducing HE proliferation was conducted during EHT induction in a RUNX1c gene reporter assay. To model human disease, dermal fibroblast-derived iPS from a hypomorphic RAG2 Omenn syndrome patient were differentiated into HSCs, and cocultured with MS5 or OP9-DL1 stromal cells to induce B or T cell development. Fluorescence-activated cell sorting (FACS) was performed weekly for 6 weeks after initiating lymphoid differentiation.Results: In the candidate screen, IFN-γ enhanced RUNX1c reporter 2.4-fold during EHT, which was confirmed by flow cytometry showing increased hematopoietic progenitor cells (CD34+CD45+). Several compounds were suppressive to EHT, including IFN-α, which abrogated EHT enhancement when combined with IFN-γ. Despite 1 week of lymphoid-directed differentiation, cell populations derived from mutant RAG2 iPS cells showed a predominance of myeloid progenitors (CD38+CD45+CD45RA+), with only 5% of progenitors manifesting a lymphoid phenotype (CD10+CD34+CD45RA+). In contrast, wildtype iPS-derived HSCs produced CD19+IgM- B cell progenitors as well as mature IgM+ B cells (8% versus .06% of CD19+CD21+ B cells), which were comparable to cord blood controls; CD3+CD4+CD8+ T cells were also identified after 3 weeks. Ultimately, we determined that RAG2 -mutant iPS were unable to generate CD19+ B cells and arrested at the early T cell stage (CD5+CD4-CD8-).Conclusion: We determined that transient exposure to IFN-γ greatly improved the EHT of HE, which may bolster the typically low yield of iPSC-derived HSCs. In a lymphoid differentiation assay, wildtype iPS-derived HE were comparable to CB in the formation of mature and immature B/T cells, whereas RAG2 -derived HEs arrested at the pro-thymocyte stage. This finding suggests that our approach to creating engraftable HSCs holds significant potential for modeling hematopoietic disease, both in animal models and as a “disease-on-a-dish,” and for developing therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Pretransplant Clinical Factors Can be Used to Predict the Risk of Cytokine Release Syndrome after Haploidentical Peripheral Blood Transplantation

Study Question: What are pre-transplant factors which predict the risk of development of Cytokine Release Syndrome after Haploidentical Peripheral Blood Transplantation?Significance: Allogeneic hematopoietic cell transplantation is a cornerstone of therapy for hematologic malignancies and often a patient's only curative intent treatment. Overall outcomes for haploidentical transplantation appear to be excellent, however, this novel approach brings toxicities that are particular to its biological and clinical milieu. We recently described occurrence of severe cytokine release syndrome (CRS) after haplo-HCT. Severe CRS was associated with poor clinical outcomes, including transplant related mortality (TRM), overall survival (OS), and neutrophil engraftment (Abboud et al, BBMT, 10/2016).However, the factors predicting the occurrence severe CRS after haplo-HCT are not known. The diagnosis of CRS requires a high level of clinical suspicion, and early treatment leads to rapid resolution of symptoms. Identifying at-risk patients prior to transplantation is clinically important.Patients and Methods: All patients (150 consecutive patients) were included who underwent peripheral blood T-Cell replete haplo-HCT with PTCy from July 2009 through October 2016 at Washington University in St. Louis. Since CRS can resemble sepsis syndrome, and additionally sepsis may be a confounding factor by causing CRS independently, patients with a documented infection or clinical suspicion of sepsis were excluded from the analysis (14 patients). Steroid use may also be a confounding variable, therefore, we excluded patients who received steroids between days -5 and the development of CRS (0 patients). Clinical data was missing for only 3 out of the 150 patients, in all cases due to random errors in institutional EMR migration, therefore they were excluded from analysis and we will not undertake imputation of missing values. CRS was graded per the criteria used in our previous work, based on the standards originally proposed by Lee et al, modified to include altered mental status and new onset cardiomyopathy.Statistical Analysis: An adaptive lasso with SBC selection with split categorical predictors was used to identify 11 predictors of severe CRS (grade 2-4). Model selection was repeated 100 times using resampled (bootstrapped) subsets of the data, and the parameter estimates averaged to obtain an average representation of predictor strength.Results: The final selection of predictors included CD34+ cells received <5.0, CD34+ cell received > 5.01, T cell dose, HLA match, CMV status of recipient, presence of a prior transplant, donor was a first degree relative, sex of donor, use of myeloablative conditioning, HCT-CI score, T cell dose and age of recipient at transplant.We stratified patients into low risk (CRS grades 0-1) or high risk (CRS grades 2-4). This matches treatment guidelines which dictate that all patients with grades 3-4, and select patients with grade 2, CRS should be treated with IL-6 inhibitors or steroids.Our predictive model had excellent specificity (94%), positive predictive value (88%), and false negative rate (6%). The sensitivity (39%), negative predictive value (58%), and false positive rate (61%) were poor.Conclusions : We have created the first model for predicting the risk of CRS in haplo-SCT using pre-transplant patient and donor characteristics. Clinically, our model proves extremely reliable to rule patients in for CRS which may require treatment. If a patient is deemed high risk by our model, they have an 88% chance of subsequently developing grade 2-4 CRS. It is less useful for ruling patients out for CRS. If a patient is deemed low risk, they still have a 42% chance of developing grade 2-4 CRS. This model is immediately clinically applicable to identify patients who must be watched very closely for CRS in the post-transplant period. It is further useful in designing CRS prevention trials, as the model can be used as an enrollment criterion to select patients at high risk for CRS who would benefit from a prophylactic approach.We are currently working with one outside institution to perform external validation of our model. We are looking for other external partners to further improve the model. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.