Our laboratory studies the basic biology and clinical utilization of NK cells. The following are the major areas of our focus:
NK cell-mediated Immunotherapy
NK and T cells hold significant promise in the formulations of novel cellular immunotherapies targeted to chemo-resistant/relapsing malignant hematopoietic and solid tumors. More importantly, recent scientific breakthroughs have helped to formulate a successful Chimeric Antigen Receptor (CAR)-based NK or T cell-mediated cellular immunotherapy. However, CAR-mediated therapy also causes a deleterious pathological condition called, 'cytokine-release syndrome' (CRS), a potentially fatal condition in two thirds of patients. CRS is a direct outcome of CAR therapy and is caused by a significantly augmented production of inflammatory cytokines by CAR-transduced lymphocytes. Two thirds of the patients infused with CAR-transduced lymphocytes develop differing levels of CRS. Formulations of novel methods that regulate inflammation are of high clinical relevance and are central to prevent CRS.
Our current work focuses on generating NK cell-based novel immunotherapies. Our clinical group headed by Dr. Monica Thakar currently offers an unmanipulated NK cell-based immunotherapy to cancer patients. She employs a cellular and adoptive immunotherapy strategy that incorporates hematopoietic cell transplantation (HCT) followed by the infusion of donor-derived NK cells into cancer patients. Clinical grade NK cells are manufactured under FDA-approved IND BB 13794 (Dr. Thakar) and infused into the patients. This therapy is being offered at the Children’s Hospital of Wisconsin and Froedtert Hospital. In addition, we are interested in defining the precise molecular mechanisms by which anti-tumor cytotoxicity and induction of inflammation are individually regulated in NK and T cells. This molecular blueprint is central to the success of the CAR-mediated clinical applications. In this context, our recent study using unmanipulated NK cells identified a unique Fyn-ADAP-Carma1 signaling pathway that is exclusively responsible for the production of inflammatory cytokines, not anti-tumor cytotoxicity (61/866,348; 8/15/2013). We are currently working to engineer a ‘CRS-free-CAR’ therapy.
Spacetime relationship of signaling events in NK cells
Spaciotemporal organization of signaling events in lymphocytes are poorly understood. Ligands initiate multiple signaling pathways via unique receptors. Hundreds of signaling molecules take part in transducing membrane proximal events into meaningful cellular functions. Although exceptional advances made in the understanding of signaling cascades, the precise mechanisms that co-ordinate and contain a pathway remain elusive. Scaffolding proteins have provided part of the explanation into how signaling events can be spatiotemporally co-ordinated. IQGAP1 is a 190 kDa cytoplasmic scaffolding protein. Here, we are working to determine how the spacetime relativity of specific signaling events is controlled by IQGAP1. We hypothesize that IQGAP1 functions as a signal processing center. Based on our preliminary work, we identify multiple major scaffolding functions for IQGAP1. First, IQGAP1 regulates β-catenin/TCF/LEF activation pathway that is involved in the terminal maturation and subset specification of NK cells. Second, IQGAP1 forms a novel signalosome around the perinuclear region to regulate ERK1/2 activation via Rac1→Pak→Raf→MEK1/2 pathway. Third, IQGAP1 plays a central role in actin polymerization, microtubule elongation and MTOC formation, which are important for the immunological synapse formation, tumor lysis and cell movement.
Results obtained from these aims will provide crucial insights into how a unique scaffolding protein regulates the development, maturation and effector functions of NK cells. These studies will also provide critical understanding of how IQGAP1 organizes membrane proximal signaling events into distal signalosomes that transiently but abundantly generate phosphorylated kinases and substrates in the perinuclear region of an effector lymphocyte.
Metabolic Reprogramming in NK Cells
NK Cells are crucial in mediating anti-tumor cytotoxicity. Transition of ‘resting’ to an ‘activated’ NK cell status requires a significant change in its bioenergetic requirements. However, the molecular mechanism that regulates this metabolic reprogramming in NK cells is yet to be defined. When and how NK cells switch to ‘Warburg’ metabolism is central to formulating successful therapeutic approaches of cancer treatment. Using two scaffold proteins, IQGAP1 and KSR1 that are predominantly expressed in lymphocytes, as molecular models we have uncovered a novel mechanism that is central to the metabolic reprogramming of NK cells. Lack of either or both IQGAP1 and KSR1 resulted in significant alteration in the ability of NK cells to switch from a primarily oxidative phosphorylation to aerobic glycolysis. Both mitochondrial function and the ability to increase the mitochondrial mass are defective in the absence of IQGAP1 and KSR1. More importantly, NK cells from Iqgap1-/-,Ksr1-/-, and Iqgap1-/-Ksr1-/- mice displayed a significantly impaired pattern of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), demonstrating an impaired mitochondrial function. In addition, lack of IQGAP1 and/or KSR1 significantly altered B-Raf/C-Raf→MEK1/2→ERK1/2→RSK1→ S6S235/S236 and PI3K-p85a→PDK1→AKT1T308→mTORC1→S6K1→S6S240/S244 signaling pathways. We hypothesize that IQGAP1 and KSR1 act as central core of a signaling complex that is indispensable for metabolic reprogramming in effector lymphocytes; therefore, their ability to mediating anti-tumor cytotoxicity and production of inflammatory cytokines/chemokines.
Results obtained through these specific aims will provide novel insights into how two scaffold proteins IQGAP1 and KSR1 regulate the metabolic reprogramming of NK cells. Since these scaffold proteins are expressed primarily in hematopoietic cells, we predict these results will be applicable to other effector lymphocytes.