Abigail Washington

Individualized vaccination of AML patients in remission is associated with induction of antileukemia immunity and prolonged remissions

A personalized DC/AML fusion cell vaccine promotes the expansion of leukemia-specific T cells and prolonged remission in patients. Immune cells join leukemia then beat it Acute myeloid leukemia (AML) is an aggressive hematologic cancer. The only curative treatment available for this disease is hematopoietic stem cell transplantation, which can result in donor immune cells helping to eradicate the cancer. Unfortunately, this procedure is not always effective and is itself associated with numerous complications and risk of death. Rosenblatt et al. have identified a potentially better way to stimulate an immune response against AML by fusing patients’ own leukemia cells with dendritic cells. The resulting fusion cells were very effective at presenting tumor antigens to T cells, resulting in a strong antitumor T cell response and prolonged survival in human patients. We developed a personalized cancer vaccine in which patient-derived acute myeloid leukemia (AML) cells are fused with autologous dendritic cells, generating a hybridoma that potently stimulates broad antitumor responses. We report results obtained from the first 17 AML patients, who achieved remission after chemotherapy and were then serially vaccinated to target minimal residual disease and prevent relapse. Vaccination was well tolerated and induced inflammatory responses at the site of administration, characterized by the dense infiltration of T cells. Vaccination was also associated with a marked rise in circulating T cells recognizing whole AML cells and leukemia-specific antigens that persisted for more than 6 months. Twelve of 17 vaccinated patients (71%; 90% confidence interval, 52 to 89%) remain alive without recurrence at a median follow-up of 57 months. The results demonstrate that personalized vaccination of AML patients in remission induces the expansion of leukemia-specific T cells and may be protective against disease relapse.

MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs

The PD-L1/PD-1 pathway is a critical component of the immunosuppressive tumor microenvironment in acute myeloid leukemia (AML), but little is known about its regulation. We investigated the role of the MUC1 oncoprotein in modulating PD-L1 expression in AML. Silencing of MUC1 in AML cell lines suppressed PD-L1 expression without a decrease in PD-L1 mRNA levels, suggesting a post-transcriptional mechanism of regulation. We identified the microRNAs miR-200c and miR-34a as key regulators of PD-L1 expression in AML. Silencing of MUC1 in AML cells led to a marked increase in miR-200c and miR-34a levels, without changes in precursor microRNA, suggesting that MUC1 might regulate microRNA-processing. MUC1 signaling decreased the expression of the microRNA-processing protein DICER, via the suppression of c-Jun activity. NanoString (Seattle, WA, USA) array of MUC1-silenced AML cells demonstrated an increase in the majority of probed microRNAs. In an immunocompetent murine AML model, targeting of MUC1 led to a significant increase in leukemia-specific T cells. In concert, targeting MUC1 signaling in human AML cells resulted in enhanced sensitivity to T-cell-mediated lysis. These findings suggest MUC1 is a critical regulator of PD-L1 expression via its effects on microRNA levels and represents a potential therapeutic target to enhance anti-tumor immunity.

MUC1 mediated induction of myeloid-derived suppressor cells in patients with acute myeloid leukemia.

Myeloid-derived suppressor cells (MDSCs) play a critical role in promoting immune tolerance and disease growth. The mechanism by which tumor cells evoke the expansion of MDSCs in acute myeloid leukemia (AML) has not been well described. We have demonstrated that patients with AML exhibit increased presence of MDSCs in their peripheral blood, in comparison with normal controls. Cytogenetic studies demonstrated that MDSCs in patients with AML may be derived from leukemic or apparently normal progenitors. Engraftment of C57BL/6 mice with TIB-49 AML led to an expansion of CD11b+ Gr1+ MDSCs in bone marrow and spleen. Coculture of the AML cell lines MOLM-4, THP-1 or primary AML cells with donor peripheral blood mononuclear cells elicited a cell contact-dependent expansion of MDSCs. MDSCs were suppressive of autologous T-cell responses as evidenced by reduced T-cell proliferation and a switch from a Th1 to a Th2 phenotype. We hypothesized that the expansion of MDSCs in AML is accomplished by tumor-derived extracellular vesicles (EVs). Using tracking studies, we demonstrated that AML EVs are taken-up myeloid progenitor cells, resulting in the selective proliferation of MDSCs in comparison with functionally competent antigen-presenting cells. The MUC1 oncoprotein was subsequently identified as the critical driver of EV-mediated MDSC expansion. MUC1 induces increased expression of c-myc in EVs that induces proliferation in the target MDSC population via downstream effects on cell cycle proteins. Moreover, we demonstrate that the microRNA miR34a acts as the regulatory mechanism by which MUC1 drives c-myc expression in AML cells and EVs.

A Phase I Study of Lenalidomide plus Chemotherapy with Mitoxantrone, Etoposide, and Cytarabine for the Reinduction of Patients with Acute Myeloid Leukemia

Patients with relapsed AML have a poor prognosis and limited responses to standard chemotherapy. Lenalidomide is an immunomodulatory drug that may modulate anti‐tumor immunity. We performed a study to evaluate the safety and tolerability of lenalidomide with mitoxantrone, etoposide and cytarabine (MEC) in relapsed/refractory AML. Adult patients with relapsed/refractory AML were eligible for this phase I dose‐escalation study. We enrolled 35 patients using a “3 + 3” design, with a 10 patient expansion cohort at the maximum tolerated dose (MTD). Lenalidomide was initially given days 1‐14 and MEC days 4‐8; due to delayed count recovery, the protocol was amended to administer lenalidomide days 1‐10. The dose of lenalidomide was then escalated starting at 5 mg/d (5‐10‐25‐50). The primary objective was tolerability and MTD determination, with secondary outcomes including overall survival (OS). The MTD of lenalidomide combined with MEC was 50 mg/d days 1‐10. Among the 35 enrolled patients, 12 achieved complete remission (CR) (34%, 90%CI 21‐50%); 30‐day mortality was 6% and 60‐day mortality 13%. The median OS for all patients was 11.5 months. Among 17 patients treated at the MTD, 7 attained CR (41%); the median OS was not reached while 12‐month OS was 61%. Following therapy with MEC and lenalidomide, patient CD4+ and CD8+ T‐cells demonstrated increased inflammatory responses to autologous tumor lysate. The combination of MEC and lenalidomide is tolerable with an RP2D of lenalidomide 50 mg/d days 1‐10, yielding encouraging response rates. Further studies are planned to explore the potential immunomodulatory effect of lenalidomide and MEC.

DCOne as an Allogeneic Cell-based Vaccine for Multiple Myeloma

Multiple myeloma (MM) is characterized by progressive immune dysregulation, loss of myeloma-specific immunity, and an immunosuppressive milieu that fosters disease growth and immune escape. Accordingly, cancer vaccines that reverse tumor-associated immune suppression represent a promising therapeutic avenue of investigation. We examined the potential of an allogeneic cellular vaccine to generate immune responses against MM tumor cells. The DCOne vaccine is comprised of a human myeloid leukemia cell line differentiated into a fully functional dendritic cell, expressing a range of tumor-associated antigens that are also known targets in MM. We found that the myeloma-specific antigens expressed by the DCOne vaccine can traffic via extracellular vesicles to surrounding antigen-presenting cells, thus stimulating autologous T-cell responses. Indeed, coculture of peripheral blood mononuclear cells from patients with MM with the DCOne vaccine resulted in the expansion of activated CD8+ T cells expressing interferon-&ggr; and perforin, with no significant change in the percentage of CD4+ T cells producing interleukin-10. Further, coculture of patient’s tumor cells with peripheral blood mononuclear cells and DCOne induced cytotoxic T-lymphocyte-mediated killing of autologous MM cells. These findings demonstrate that the allogeneic DCOne vaccine can induce T-cell activation and myeloma-specific immunity via cross presentation of antigens by native antigen-presenting cells.

MUC1‐C drives myeloid leukaemogenesis and resistance to treatment by a survivin‐mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy‐resistant disease. The MUC1‐C oncoprotein governs critical pathways of tumorigenesis, including self‐renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1‐C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1‐C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1‐C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1‐C with silencing or pharmacologic inhibition with GO‐203 led to a decrease in active β‐catenin levels and, in‐turn, down‐regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1‐C was also associated with increased sensitivity of AML cells to Cytarabine (Ara‐C) treatment by a survivin‐dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1‐C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO‐203 with Ara‐C for the treatment of patients with AML.

Phase 1 clinical trial evaluating abatacept in patients with steroid-refractory chronic graft-versus-host disease

Steroid-refractory chronic graft-versus-host disease (SR-cGVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. Innovative immunotherapeutic strategies are urgently needed for the treatment of SR-cGVHD. We conducted a phase 1 clinical trial to evaluate the safety, efficacy, and immune effects of abatacept, a novel immunomodulatory drug that acts as an inhibitor of T-cell activation via costimulatory blockade, in the treatment of SR-cGVHD. The study followed a 3+3 design with 2 escalating abatacept doses: 3 mg/kg and 10 mg/kg, with an expansion cohort treated at 10 mg/kg. Abatacept was well-tolerated with no dose-limiting toxicities. Of the 16 evaluable patients, 44% achieved a clinical partial response per 2005 National Institutes of Health Consensus Criteria. Importantly, abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders (mean baseline, 27 vs 14 mg; P = .01). Increased PD-1 expression on circulating CD4 (P = .009) and CD8 (P = .007) T cells was observed in clinical responders. In summary, abatacept was safe and led to a marked improvement in National Institutes of Health cGVHD scores and a significant reduction in prednisone use. In this cohort of heavily pretreated patients, the results suggest abatacept may be a promising therapeutic agent for SR-cGVHD, and a phase 2 trial has been initiated. This trial was registered at www.clinicaltrials.gov as #NCT01954979.

Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous neoplasm and patients with relapsed/refractory disease exhibit resistance to standard therapies. We have previously demonstrated that the Mucin 1 C-terminal subunit (MUC1-C) plays a critical role in protection from oxidative stress in CTCL cells. Targeting of MUC1-C with a pharmacologic inhibitor, GO-203, was associated with apoptosis in CTCL. However, disease responses were incomplete underscoring the need for combinatorial strategies that could exploit the vulnerability of CTCL cells to oxidative signals. Cell lines, primary samples, and xenograft models of CTCL were used to assess synergy of GO-203 with decitabine, a hypomethylating agent. Present studies demonstrate that exposure of CTCL cells to decitabine in combination with GO-203, increased the generation of reactive oxygen species (ROS) levels and decreased levels of scavenger molecules, NADP, NADPH, glutathione, and TIGAR, critical to intracellular redox homeostasis. Dual exposure to GO-203 and decitabine resulted in marked downregulation of DNA methyl transferases demonstrating significant synergy of these agents in inducing global and gene specific hypomethylation. Accordingly, treatment with decitabine and GO-203 upregulated the ROS generating enzymes, NADPH oxidase 4 and dual oxidase 2 potentially due to their effect on epigenomic regulation of these proteins. In concert with these findings, exposure to decitabine and GO-203 resulted in heightened apoptotic death in CTCL cell lines, patient-derived primary samples and in a murine xenograft model. These findings indicate that decitabine intensifies MUC1-C inhibition induced redox imbalance and provides a novel combination of targeted and epigenetic agents for patients with CTCL. Mol Cancer Ther; 16(10); 2304–14. ©2017 AACR.

Abstract 19: Hypomethylating agent, SGI-110, alters the immunosuppressive milieu in acute myeloid leukemia (AML) and enhances the immunogenicity of a dendritic cell/AML fusion vaccine

Acute myeloid leukemia (AML) is associated with an immunosuppressive tumor microenvironment (TME) that prevents immune recognition and fosters disease growth. In an attempt to reverse this immune suppression, we developed a personalized vaccine in which patient-derived AML cells are fused with autologous dendritic cells (DCs) such that a broad array of leukemic antigens (Ags) is presented in the context of DC-mediated costimulation. We have examined strategies to enhance vaccine efficacy. In this regard, we previously demonstrated that the hypomethylating agent (HMA), SGI-110, augments leukemic Ag processing and increases AML cell susceptibility to T cell-mediated killing. In the present study, we investigated if SGI-110 enhances leukemic Ag presentation by measuring the binding capacity of a unique T-cell receptor-like (TCRL) antibody (Ab) to AML cells in vitro. The TCRL Ab is a TCR mimic that recognizes the PR1-HLA-A2-MHC complex on the surface of leukemic cells where PR-1 is a known leukemic Ag. Following exposure of SGI-110 to the AML cell line, THP-1 which endogenously expresses HLA-A2, we noted enhanced tumor Ag presentation and HLA-A2 upregulation via FACS analysis. Aside from facilitating Ag presentation, we investigated if SGI-110 affects myeloid-derived suppressor cell (MDSC) burden in the TME where MDSCs have immunosuppressive capabilities. In an in vitro assay where healthy donor-derived peripheral blood mononuclear cells were cocultured with 2 AML cell lines, SGI-110 treatment led to a significant decrease in MDSC expansion compared to control. Based on these in vitro data, we investigated the ability of SGI-110 to reverse critical aspects of the immunosuppressive milieu in AML and enhance vaccine efficacy in vivo. C57BL/6J mice were retro-orbitally inoculated with 1x10 5 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1 mg/kg) to assess immunologic effects while not preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). Similarly, SGI-110 therapy was associated with a decrease in MDSC burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control mice. Consistent with these findings, SGI-110-treated mice demonstrated increased AML-specific immunity as manifested by increased T cells isolated from the BM or spleen that expressed intracellular IFNγ following ex vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02). We subsequently examined the potential synergy of SGI-110 plus the vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNγ in response to tumor lysate as compared to vaccine or SGI-110 alone. Given these effects, we sought to examine whether treatment with the vaccine and SGI-110 would enhance survival in this model. Mice were treated with vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a statistically significant survival advantage as compared to untreated mice; yet the combination arm further prolonged survival as compared to either monotherapy arm. In conclusion, SGI-110 enhances Ag presentation and modulates the AML-associated immunosuppressive milieu by decreasing both T-cell PD-1 expression and MDSC burden with a notable increase in AML-specific immunity. Moreover, SGI-110 increases the immunogenicity of the DC/AML vaccine through an enhanced AML-specific response with a survival benefit in a murine model. This combination strategy has promising translational potential and a phase 1 clinical trial with this synergistic approach is planned. Citation Format: Myrna Rita Nahas, Dina Stroopinksy, Anna Sergeeva, Athalia Pyzer, Abigail Washington, Leandra Cole, Salvia Jain, Malgorzata McMasters, James D. Levine, Rebecca Karp Leaf, Shira Orr, Matthew Weinstock, Jeffrey J. Molldrem, David Avigan, Jacalyn Rosenblatt. Hypomethylating agent, SGI-110, alters the immunosuppressive milieu in acute myeloid leukemia (AML) and enhances the immunogenicity of a dendritic cell/AML fusion vaccine [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 19.