Todd A. Fehniger, Ph.D. - US grants

DESCRIPTION (provided by applicant): The long term goal of this proposal is to train the applicant to become an independent academic physician- scientist studying the innate immune system and its impact on blood cancers. The principal investigator (PI) has completed PhD training focused on the cellular biology of natural killer (NK) cells, and MD training in internal medicine and hematology-oncology. This application describes a 5 year training program that will provide a mentored educational experience aimed at developing new scientific expertise in molecular profiling, massively parallel sequencing, proteomic analysis, manipulation of microRNAs (miRs), and the generation of mouse models. Dr. Timothy Ley will mentor the Pi's scientific and career development. He is a recognized leader in the field of lymphocyte cytotoxicity, in vivo genetic mouse models, and genomic analysis of leukemia. Furthermore, an advisory committee of medical scientist experts will provide additional scientific and and non-scientific career development guidance. The proposed research will evaluate the role of miRs in the regulation of NK cell cytokine activation. Recent work by the PI in Dr. Ley's laboratory identified two critical cytotoxic molecules, granzyme B (GzmB) and perforin (Prf1), that are post-transcriptionally regulated in NK cells. We hypothesize that miRs regulate GzmB and Prf 1 in resting NK cells, and that cytokine-activation releases their block in translation. To address this hypothesis, we propose the following specific aims: 1) We will define the miR expression profiles in resting and cytokine-activated NK cells, and evaluate candidate miRs that may regulate GzmB and Prfl mRNA translation. 2) We will define the mRNA (transcriptome) and protein (proteome) expression profiles of resting and cytokine-activated NK cells, integrate these databases to define the mode of regulation of molecules important during NK cell activation, and define the role of miRs for post-transcriptional regulation. Techniques utilized in the project include miR sequencing, miR microarrays, in vitro and in vivo manipulation of miRs, the generation of a NK cell-specific Cre mouse model, the generation of genetic mouse models deficient in miRs using Cre-Lox, and the global analysis of the NK cell transcriptome and proteome. Washington University provides an ideal setting to train physician-scientists, and will foster an invaluable mentored educational experience for the PI to realize his career goals in academic medicine. This overall career development proposal will train an independent physician-scientist for a lifetime of research studying the immune system and cancer. As NK cells are key components of immunity to numerous infectious pathogens, and are involved in the immuno-surveillance of malignancy, the research proposed may have far reaching consequences for health and disease. Specifically, a better understanding of NK cell activation may lead to novel immune based strategies to treat hematologic malignancies.

DESCRIPTION (provided by applicant): The long term goals of this application are to define molecular events that regulate NK cell development and function, thereby providing insight into pathways that may be manipulated to promote or inhibit these aspects of NK cell biology. NK cells are innate immune lymphocytes that are important during host defense against infections and mediate anti-neoplastic immune responses. This is exemplified by rare patients with selective NK cell deficiencies that succumb to fatal herpesvirus infections early in life, demonstrating the critical importance of these cells for human health. Moreover, allogeneic NK cells can be an effective treatment for acute myeloid leukemia patients, and thus NK cells hold promise as a cancer immunotherapy approach. Currently, we have an incomplete understanding of the molecular mechanisms responsible for regulating NK cell functionality (cytokine production, cytotoxicity, proliferation). MicroRNAs (miRNAs) are small regulatory RNAs that target mRNA stability and/or limit protein translation, consequently regulating critical cellular processes. Recent studies have identified miRNAs expressed in resting and activated mouse and human NK cells, and have shown that global miRNA deficiency results in altered mature NK cell functional responses in vitro and in vivo. However, our understanding of how individual miRNAs regulate NK cell biology is limited. Of multiple candidate miRNAs that are expressed and have a rationale for regulating NK cell function, miR-155 was prioritized at the highest for in depth study. This application describes a 5 year plan to study the regulatory role o miR-155 on NK cell function. We hypothesize that miR-155 regulates key aspects of NK cell functionality, including effector cytokine (e.g., IFN-?) production, cytotoxicity, and intracellula pathways important for promoting or inhibiting NK cell activation. Our approach utilizes NK cell-specific (Ncr1-iCre) conditional gain (via loxP-STOP-loxP overexpression) and loss (floxed) of function models for miR-155. In Aim 1 miR-155 mouse models will be used to define the regulatory contribution of miR-155 to NK cell function, including development/maturation, IFN-? production, and cytotoxicity. In Aim 2 we will elucidate the regulatory mechanism(s) of miR-155 by defining their target mRNAs in NK cells using bioinformatics, mRNA profiling, and Argonaute-immunoprecipitation followed by next-generation sequencing. These targets will in turn be assessed in functional experiments, described in Aims 1 and 3, and confirmed in human NK cells. In Aim 3 we will define the in vivo significance of miR-155 regulation on NK cell function using model pathogens. There is a high level of integration between Aim 2 and Aims 1 and 3, to deeply define mechanisms whereby miR-155 targets specific mRNAs, which in turn regulate NK cell function. Thus, these studies will provide novel information about how miR-155 regulates NK cell activation, their mechanism of action by identifying mRNA targets, and the importance of miR-155 to NK cell-mediated host defense in vivo. These studies may therefore inform future strategies to augment NK cell functionality as an immunotherapeutic strategy.

The long term goal of this project is to translate novel findings in the field of innate immunity into early phase immunotherapy clinical trials for patients with hematologic malignancies. In this proposal we focus on acute myeloid leukemia (AML), an aggressive cancer of developing myeloid cells that has a poor prognosis, with <30% of patients treated with standard therapy achieving long-term disease-free survival. While allogeneic hematopoietic cell transplantation (HCT) is a standard treatment that is potentially curative for some patients with AML, this therapy is associated with significant morbidity (graft versus host disease and infection) and treatment-related mortality. This limits HCT applicability and overall effectiveness in this disease of older individuals. One promising strategy that preserves the anti-leukemia effector function against AML, without the morbidity and mortality of HCT, is the adoptive transfer of allogeneic lymphocytes. Natural killer (NK) cells are innate lymphoid cells that are specialized to eliminate malignantly transformed target cells. Clinical studies for AML patients using HLA-haploidentical allogeneic HCT have shown that the reactivity of donor NK cells against the patients' leukemia predicts for long-term disease-free survival. Adoptive immunotherapy with enriched allogeneic NK cell products administered to patients with active AML have resulted in complete remissions, although these are achieved in a minority of patients and are of limited duration. We hypothesize that enhancing NK cell recognition of, and effector function against, AML blasts will result in improved clinical outcomes following adoptive NK cell therapy. Recently, paradigm shifting studies have shown that NK cells exhibit immune memory, a property previously attributed only to adaptive T and B lymphocytes. We have established that human NK cells exhibit innate memory following a brief combined stimulation with interleukins (IL)-12, -15, and -18. Preliminary data demonstrates that memory-like NK cells exhibit significantly enhanced AML recognition, functionality, longevity, and proliferative potential compared to naive or control NK cells. Recent preliminary data also shows that administration of allogeneic memory-like NK cells is safe, feasible, and results in clinical responses in AML patients. Thus, we hypothesize that allogeneic memory-like NK cells administered as adoptive immunotherapy for patients with AML will exhibit potent anti-leukemia responses. In this proposal, we will 1) test the safety and efficacy of allogeneic memory-like NK cell adoptive immunotherapy in a first-in-human phase 1/2 clinical trial for patients with relapsed AML, 2) define memory-like NK cell correlates of clinical response, and elucidate key mechanisms important for memory-like NK cell anti-AML responses, and 3) define the importance of NKG2A as a memory-like NK cell checkpoint, and elucidate mechanisms of AML resistance to memory-like NK cell therapy.

Project Summary/Abstract The long-term goals of this project are to translate novel findings in the field of immunology into early phase immunotherapy clinical trials for patients with leukemia. Allogeneic hematopoietic cell transplantation (HCT) is a standard treatment for high-risk or relapsed AML and is potentially curative. However, major barriers to success in older AML patients include 1) an inability to tolerate intensive conditioning, 2) disease relapse, 3) graft-versus-host disease (GVHD), and 4) the lack of a suitable donor in many cases. One key immune player in mediating the graft-versus-leukemia (GvL) effect that also potentially limits GVHD is the NK cell. We hypothesize that new findings in NK cell biology can be translated to the clinic to improve the effectiveness and tolerability of HCT in older AML patients. Recent clinical studies have reported that HCT from an MHC-haploidentical donor (haplo-HCT) with myeloablative conditioning regimens that incorporate post-HCT cyclophosphamide results in clinical outcomes that are comparable to HCT from matched unrelated donors. However, one major obstacle to treating AML patients with haplo-HCT is the large number of older patients that are only candidates for reduced-intensity conditioning (RIC), which results in lower treatment-related mortality, but at the cost of a much higher incidence of AML relapse, and thus far, poor long-term disease-free survival. To address this important hurdle in the field, we will augment HCT with same-donor memory-like NK cell adoptive immunotherapy during the immediate post-HCT period, to enhance GvL while potentially improving engraftment and minimizing GVHD. Reports have recently identified that NK cells exhibit ?memory-like? properties following combined cytokine pre-activation. We and others have established that human memory-like NK cells respond robustly after a second stimulation and have multiple anti-tumor properties. We have translated this into a cellular therapy for relapsed/refractory (rel/ref) AML patients, and have completed a phase 1 study. However, one drawback of this allogeneic NK cell therapy is its rejection by the recipient's recovering immune system after 2-3 weeks, providing a short ?window of opportunity? for these NK cells to eliminate AML. To address this limitation in the NK immunotherapy field, we will incorporate a donor-matched RIC HCT, providing an ideal immune-compatible environment for memory-like NK cells to expand and attack residual AML. In this proposal, we will 1) test the safety and efficacy of augmenting RIC HCT with same-donor memory- like NK cell adoptive immunotherapy in a phase 2 clinical trial for patients with AML, 2) define memory-like NK cell correlates of clinical response, and elucidate key mechanisms important for memory-like NK cell anti-AML responses. These studies will lead to a new understanding of mechanisms whereby NK cells effectively attack AML, and whereby AML resists NK cell therapy, and hence strategies to improve memory-like NK cell anti- AML responses in future clinical trials.