Michael H. Overholtzer, Ph.D. - US grants

DESCRIPTION (provided by applicant): Recently a mechanism was described whereby human cells internalize into neighboring cells, called entosis. Entosis underlies the formation of 'cell-in-cell' structures, where viable cells are engulfed inside of others. These unusual cell structures have been reported in human tumors for decades, but their physiological role remains unknown. Entosis is induced by detachment of cells from extracellular matrix in vitro, and is prevalent in anchorage-independent growth assays in soft agar. In breast tumors, cell-in- cell structures are found in early-stage (DCIS) tumors, and also in late stage invasive tumors, in matrix-deprived regions, suggesting that this process could affect the formation or metastatic spread of cancers. Although cells internalized by entosis are initially viable, most eventually undergo cell death, suggesting that entosis could be a mechanism of tumor suppression. Cell death occurs by a nonapoptotic mechanism that can eliminate cells which are resistant to apoptosis. Entosis may therefore act as a backup or cooperative tumor suppressive mechanism to apoptosis to prevent transformed growth. The identification of this cellular program, whose evidence in vivo far predates the in vitro mechanism, was made possible only by real-time imaging of a classical assay of tumorigenicity, where the basic cellular programs that control the ability to grow are not defined. This proposal describes plans to examine tumorigenic transformation by real-time imaging, to elucidate the molecular mechanisms of entosis, and to examine the role of this process in human cancers.

DESCRIPTION (provided by applicant): The end stages of the cellular engulfment mechanisms phagocytosis and entosis, which mediate the uptake of exogenous substrates, and autophagy, which mediates the engulfment of intracellular substrates, involve lysosomal digestion of engulfed cargo and export of catabolites to the cytosol for use in biosynthesis. Despite the critical role of these pathways for the removal of targeted substrates, and the importance lysosome- mediated digestion for the actual clearance and recycling of engulfed material, little is known about how the lysosomal processing of engulfed cargo and export of degraded components is regulated. We have found that the mTOR protein kinase, a regulator of mRNA translation and autophagy, is required for a program of phagosome fission that shrinks large lysosomal vacuoles as internalized cargo is degraded, which reminiscent of the recently described autophagic lysosome reformation (ALR) program that functions similarly during autophagy. mTORC1 is activated by amino acid recovery from engulfed cells, and is recruited specifically to large lysosomal vacuoles harboring degrading cells to control their fission. The proposed research will identify amino acid transporters acting at lysosomes that mediate amino acid recovery and mTORC1 activation, and will identify mTOR-regulated proteins that control the fission of lysosomal vacuoles.

DESCRIPTION (provided by applicant): The end stages of the cellular engulfment mechanisms phagocytosis and entosis, which mediate the uptake of exogenous substrates, and autophagy, which mediates the engulfment of intracellular substrates, involve lysosomal digestion of engulfed cargo and export of catabolites to the cytosol for use in biosynthesis. Despite the critical role of these pathways for the removal of targeted substrates, and the importance lysosome- mediated digestion for the actual clearance and recycling of engulfed material, little is known about how the lysosomal processing of engulfed cargo and export of degraded components is regulated. We have found that the mTORC1 protein kinase, a regulator of mRNA translation and autophagy, and the lipid kinase PIKfyve, are required for a program of phagosome fission that shrinks large lysosomal vacuoles as internalized cargo is degraded, which reminiscent of the recently described autophagic lysosome reformation (ALR) program that functions similarly during autophagy. Vacuole fission is associated with nutrient recovery that rescues engulfing cells from the effects of amino acid or glucose/pyruvate starvation, and reactivates mTORC1. Reactivated mTOR recruits specifically to large lysosomal vacuoles harboring degrading cells and controls their fission. The proposed research will identify amino acid and sugar transporters acting at lysosomes that mediate nutrient recovery and mTORC1 activation, and will identify mTOR and PIKfyve-regulated proteins that control the fission of lysosomal vacuoles.

Project Summary: Enormous strides continue to be made in the design of nanoparticles as highly specialized therapeutics for achieving superior outcomes over standard pharmacological agents, the latter often associated with significant toxicity that limits treatment efficacy. While cancer immunotherapies have revolutionized the treatment of disease and shown therapeutic benefits in hard-to-treat cancers, these agents are limited, for example, by immune-related adverse events and off-target effects in immunosuppressive microenvironments. Novel, emerging anti-cancer strategies are therefore critically needed to overcome these limitations and improve durable response rates in combination with immune therapies. One promising strategy exploits the unique ?self- therapeutic? capabilities of the nanomaterials themselves ? the treatment of tumors without the need for cytotoxic drugs. These capabilities are governed by the intrinsic physico-chemical properties of these materials, which can lead to disruption of signal transduction pathways, cell cross-talk or invasion, and/or induced cell death programs within the tumor microenvironment (TME) ? providing unprecedented opportunities for combating disease. We have developed specialized ultrasmall fluorescent core-shell silica nanoparticles, Cornell prime dots (C' dots), with intrinsic therapeutic capabilities enabling a distinct combination of activities that (1) selectively and directly induce cancer cell death through the iron-dependent mechanism of ferroptosis and (2) modulate immune cells directly by priming T cells and polarizing macrophages toward a pro-inflammatory phenotype. As CD8+ T cells are known to also regulate ferroptosis during immunotherapy, such effects are expected to synergize with those induced by C' dots. A long-term goal of this proposal is to determine critical C' dot physico-chemical parameters responsible for maximizing responses to these intrinsic therapeutic activities. In Aim I, we will examine the extent to which changes in the structural properties of PEG-coated C' dots, plain or modified to specifically bind to melanocortin-1 receptor (MC1-R; a well-established target overexpressed by our syngeneic murine models and human melanomas), influence therapeutic efficacy in syngeneic melanoma models by modulating ferroptosis and the tumor microenvironment, in the presence and absence of checkpoint blockade. In Aim II, we will probe underlying mechanisms driving regulation of immune cell phenotype and/or induction of ferroptosis in vitro. The successful completion of the project will provide critical insights into (i) key structural parameters modulating the combined self-therapeutic activities of these particles related to their induction of ferroptosis and priming the tumor immune microenvironment; (ii) whether critical differences exist in particle characteristics needed to optimize these distinct activities; (iii) mechanisms underpinning these activities; and (iv) therapeutic strategies that maximize potent anti-tumor effects in syngeneic melanoma models by administering therapeutic doses of particles in tandem with checkpoint inhibitors (anti-PD-1 and anti-CTLA-4).

PROJECT ABSTRACT The central mission of the T32 ?Molecular Imaging in Cancer Biology? program (T32 MICB) will be to develop novel molecular imaging methods, technologies, and platforms that will accelerate the understanding of human cancer biology as a basis for designing curative cancer therapeutic regimens as well as cutting-edge diagnostic, prognostic, and therapeutic tools. This overall goal will be achieved under the auspices of three key themes: (1) ?Imaging Fundamental Biology,? (2) ?Imaging New Model Systems,? and (3) ?Imaging Technology Development.? This T32 MICB research portfolio is situated at the intersection of various disciplines?from basic science including chemistry and physics, structural biology, drug development, cell biology, and developmental biology to cancer biology, invasion and metastasis, and applied disciplines, including pharmacology, nanotechnology, radiochemistry, engineering, and medicine. Fundamental knowledge about the biology of cancer has burgeoned, but the translation of basic science discoveries to clinical advancements can be slow and inefficient; molecular imaging can significantly accelerate this process and a well-trained population of basic scientists well-versed in all aspects of molecular imaging is crucial to the success of this endeavor. The faculty preceptors chosen for this T32 MICB reflect the broad range of expertise and are leaders in their respective imaging fields. They are well-funded and have independent R01 (cancer-related) or R01-like research support (e.g., R35 or HHMI). To ensure that the trainees (five post-doctoral slots and one pre-doctoral slot per year) have a full well-rounded training experience (including resilience and well-being) in preparation for independent careers, our initial design is based on a three-year training program for both the postdoctoral fellows and the predoctoral graduate student trainees. Prior to entry into the T32 MICB program and during the program, the trainees will be informed about the mission of the T32 MICB program, which is to produce scientists with specialized knowledge in advances in cancer biology and imaging technology, within a culture of respect and engagement. Furthermore, trainees will be informed about expected achievements during the program and that their progress will be continuously evaluated and benchmarked.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.