MOROSS INTEGRATED CANCER CENTER GRANTS

Regulatory T cells (Tregs) are a critical component of solid tumors, contributing to an immunosuppressive tumor microenvironment (TME). A comprehensive understanding of the mechanisms by which Tregs shape the TME and the factors influencing responses to Treg-targeted therapies remains elusive. Our research focuses on deciphering the complex interactions within the TME and understanding how Tregs influence its spatial organization. In this POC study, we aim to establish methodologies to uncover the intricate web of immune interactions within the TME. Leveraging the PhenoCycler-Fusion System (CODEX), we will perform a spatial characterization of the immune TME of two distinct tumor models, with varying sensitivities to Treg-mediated suppression. Subsequently, we will investigate the impact of Tregs on the spatial organization of the TME. By employing inducible genetic mouse models and Treg-depleting monoclonal antibodies, we will elucidate how Treg elimination influences the immune composition, pathways, and interactions within the TME. This research has the potential to significantly enhance our knowledge of immune surveillance mechanisms and the spatial dynamics of the TME. It may offer new insights into the mechanisms of Treg-mediated tumor immune suppression, potentially optimizing Tregtargeted therapies. Furthermore, we anticipate identifying specific immune cell interactions that could serve as valuable therapeutic targets, paving the way for the development of novel immune cell-based therapies to enhance anti-tumor immunity.

Immunotherapy has sparked new hope for oncology due to its remarkable ability to induce a durable
response in patients with metastatic cancer. However, a large fraction of patients do not respond to
immunotherapy. The immune response against tumor cells is driven by neoantigen presentation. Yet, most
tumors harbor a low mutational burden, limiting their targetable neo-peptidomic space. We propose to
address the urgent need to expand the search for neoantigens, by focusing on neopeptides derived from
cancer-specific translation aberrations, an avenue we recently pioneered. We demonstrated that Tryptophan
depletion evokes protein synthesis events that lead to the presentation of immunogenic aberrant peptides.
Moreover, we found an additional class of tumor-presented antigens that are derived from intra-tumor
bacteria that could be used as potential immunotherapy targets.
In this proposal, we suggest utilizing bacteria to deplete tryptophan in the tumor microenvironment by
engineering bacteria that colonize melanoma tumors to overexpress tryptophan-depleting enzymes. We will
assess whether the engineered bacteria induce aberrant peptide presentation by mouse melanoma cells. We
will test in vivo whether the modified bacteria can reach and colonize tumors and cause localized tryptophan
depletion. We will further test how this depletion affects tumor growth and response to checkpoint
inhibitors. Lastly, we will identify presented aberrant peptides in vivo, test the peptides' immunogenicity,
and design mRNA vaccines targeting these peptides. The proposed studies have significant translational
implications, with the potential to identify novel strategies to enhance immunotherapy modalities applicable
to a large number of patients with various tumor mutational burdens.

Colorectal cancer (CRC) constitutes one of the leading causes of cancer-related deaths worldwide, which
typically insidiously develops through a long years-long process involving mutations accumulating in
different genes that are essential to a variety of cellular pathways such as WNT, RAS?MAPK, TGF-?, P53,
and DNA mismatch-repair pathways. The gut microbiome has been suggested to causally impact CRC
development, progression, and response to treatment. However, the mechanisms by which gut microbial
signals impact intestinal epithelial cell signaling and transformation remain elusive. Such causal
understanding of host-microbiome modulatory activity on signaling pathways central to CRC development
and clinical behavior may hold promise in harnessing microbes and their secreted products in preventing and
treating CRC. In this proposed project, we aim to establish a high throughput screening pipeline of live
imaging to quantify the simultaneous changes in the major signaling pathways. This will enable us to
massively screen thousands of microbiome-related, food-related and host-related molecules involved in the
host-microbiome niche and their simultaneous effects on multiple key signaling pathways in human CRC.
Top reproducible hits will then be validated on their effects in mouse and human organoid cultures, and then
in in vivo setting in models of CRC in mice (such as AOM/DSS and Apcmin/+). Together, this in vivo to in
vitro to in vivo approach will help in better understanding the host-microbiome modulatory activity on
signaling pathways central to CRC development and clinical behavior, which may hold promise in
harnessing microbes and their secreted products in preventing CRC.

Malignant tumors present a distinct challenge to the immune system as "altered self". Although there is a well-established crosstalk between the immune system and the tumor that has substantial implications for cancer therapy, the nature of the antigens that allow the immune system to distinguish cancer cells from non-cancer cells has long remained obscure. Recent findings provide an important scientific linkage between the cancer immunology and autoimmunity fields and suggest that targeted breakdown of self-tolerance to various tissue-specific antigens (which are highly expressed by the corresponding tumor) could potentially be exploited as the "magic bullet" to ultimately defeat cancer. It is well established that Aire-deficient mice on NOD genetic background develop severe autoimmune phenotype, characterized by generation of a plethora of autoantibodies against multiple tissues. Therefore, in this research project we aim at exploiting breakdown of immunological tolerance to hundreds of tumor-specific antigens, which occurs naturally in AIRE-deficient mice for cancer immunotherapy. Specifically, we aim at isolating autoantibodies from AIRE-deficient mice, targeting extracellular domains of various membrane-bound antigens associated with various solid tumors, including liver (ASGR1), ovarian (MUC16) and urinary bladder (UPK2) tumors. To this end, we build on our promising preliminary data and experimental platforms we have recently developed for isolation, cloning humanization and expression of autoantibodies against the desired targets. We have identified and isolated several promising autoantibody clones to the above tumor-associated antigens and are in the process of their validation and characterization. The most promising clones will then be used for generation of antibody drug conjugates (ADC), which will then be tested for their tumor cytotoxic capacity in both in vitro and in-vivo mouse xenograft models. So far, we have successfully generated and tested ADC derived from 2B5 mAb clones that specifically targets a unique epitope of MUC16. We hope to generate ADCs for ASGR1 and UPK2 in the near future.

Liquid biopsy is a promising non-invasive approach for early detection of cancer and monitoring disease progression and therapeutic response. We have recently developed a powerful and reliable method to isolate circulating small extracellular vesicles (sEVs) from plasma of breast cancer patients and showed that semi-quantitative proteomic profiling of sEVs can be used for early detection of breast cancer. Our preliminary analysis also suggests that the sEVs proteome could be used for prediction of recurrent disease.  In this proof-of-concept project, we proposed not only to improve our isolation and profiling methods of circulating sEVs, but also to identify possible connections between the transcriptome of the primary breast tumors and the proteome of the circulating sEVs.  To this end, we plan to profile matching samples of sEVs and primary tumors from breast cancer patients and to establish prediction models by machine learning as well as connectivity map between the tumor transcriptome and the sEVs proteome using computational methods. Deconvolution models will be applied to stratify sEVs based on their cells of origin. This combined analysis may predict sEVs biomarkers for distinct properties of the primary tumors, including tumor stage, tumor composition, heterogeneity, and response to treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, often diagnosed in advanced and inoperable stage. Diagnosis requires histologic confirmation, typically obtained through endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP). These procedures are invasive and pose significant risks to patients. Recent discoveries in our lab have shown that shed cells from luminal washes carry information about their tissue of origin. This insight surpasses traditional biopsies in detecting conditions like inflammatory bowel disease (IBD). This project aims to develop a new, less-invasive diagnostic approach for PDAC diagnosis. We plan to use the transcriptomic profiles of shed cells to identify PDAC, thus improving patient care by reducing the invasiveness of the diagnostic process.

In this study we found that localized delivery of a type I Interferon (IFNβ) to a severe mouse melanoma model (B16F10) by injecting an adeno-associated-virus (AAV) as delivery system directly into the tumor we sensitize animals to respond favorably to anti-PDL1 immunotherapy. Localized AAV-IFNβ delivery results in enrichment of immune cells in the tumor microenvironment.  This may supplement immune recognition of tumors by IFNg, which we show to act synergistically with IFNb.  Interestingly, we show that the transcription factor Interferon-Regulatory-Factor 1 (IRF1) mimics the activation by IFNg, and IFNb. This even in the absence of a functional interferon system.  These novel findings open the possibility for a new melanoma treatment regime, which combines the activities of IFNg, IFNb and IRF1 for as a more potent melanoma treatment.

B cells and B cell lymphoma depend on BCR signaling for their survival. Removal or inhibition of the immune receptor signaling lead to elimination of the malignant cell. Thus, immune-receptor signaling pathways are an excellent target for cancer treatment. We aim to establish a large-scale screening method that generates numerous readouts in response to hundreds of perturbations by combing high throughput screening with CyTOF readouts.  This would allow to define the effect of each treatment on immune cells and lymphoma cells from patients and examine multiple parameters readout by CyTOF.

We hypothesize that mechanisms underlying cell size regulation in normal cells are diverted to survival/death pathways in cancer cells, raising the prospect of selectively targeting such mechanisms as a therapeutic strategy. Identifying key hubs in intracellular networks that may shift a size-sensing mechanism to control of proliferation or survival is likely to open new avenues for therapeutic development in the future.

Cancer cells tend to acidify their immediate microenvironment due to increased consumption of glucose at limiting oxygen conditions. These unique biophysical properties could be utilized to achieve differential recognition of antigens using the same therapeutic mAbs. Thus, incorporating pH-switchable recognition elements into therapeutic antibodies could result with the desired differential recognition. This would allow increasing the dose of the mAbs to maximize the therapeutic effect while reducing the severity of, or completely eliminate the off-target side effects.

Multiple types of circulating tumor cells (CTCs) metastasize into the lung parenchyma by extravasating the pulmonary vasculature. The space between the alveolus and the capillary (approximately 2 um) imposes a significant mechanical barrier on extravasating CTCs that need to squeeze their bulky and stiff nuclei through it with minimal nuclear damage. Using live imaging of the lung parenchyma with 2-photon microscopy set-up integrated with a thoracic window apparatus we will dissect the mechanisms by which cells transiently reduces their nucleus stiffening enabling extravasation. Genetic manipulations of several breast cancer cell lines by transient and permanent lamin A/C silencing and by CRISPR technology to eliminate a key laminin serine phosphorylation site will be utilized to unravel molecular players involved.

Immunotherapy has sparked new hope for oncology due to its remarkable ability to induce durable clinical
responses in cancer patients. A great potential pool of immune-stimulating factors are the neo-peptides, the
degradation products of altered proteins in cancerous cells. Yet, the targetable pool of neopeptides encoded
by mutations is limited by the low mutational burden of most tumors. Thus, strategies to increase the
neopeptide burden of tumors offer the potential of enhancing tumor immunogenicity. Building on pioneering
work by our group (Nature 2021), we propose to address this challenge via development of small molecule
inhibitors allowing to perturb tRNA modifying enzymes, which are critical for translational fidelity. In
critical, proof-of-concept experiments, we recently silenced TYW2, a tRNA-modifying enzyme, which led
to induction of aberrant, highly immunogenic peptides, giving rise to tumor growth inhibition in vivo. In this
proposal we have three goals: First, to identify small molecule TYW2 inhibitors to trigger anticancer
immunity (Aim 1). Second, we will conduct a systematic CRISPR-based screen for frameshift suppressing
tRNA-modifying enzymes. Third, we will assess the anti-tumoral potential of tRNA modifying enzyme
inhibitors, prioritized from Aim 2. Our preliminary results strongly support the feasibility of our approach
across all three aims. Identifying translation fidelity-derived neopeptides requires a deep understanding of
the cancerous translation machinery and the development of innovative experimental and computational
strategies. Our multi-disciplinary team's strengths and prior experience in tumor immunology and
(Samuels), tRNA biology (Schwartz) and medicinal chemistry (London) makes it ideally suited to fulfill
these requirements.

Tumor-targeting antibodies or reactivation of immune cells by immune checkpoint inhibition have added new treatment strategies in various types of cancer patients. In preliminary experiments, we found antibody-forming cells in NSCLC patients with tumor-binding capacity. Here, we will examine the specificity of antibodies to tumors, comprehensively characterize them, and test their therapeutic potential. Specifically, we will express antibodies recovered from the patients in different isotype formats, compare their tumor binding capacity and examine binding to tumor targets and endogenous viral associated targets. We will structurally characterize the mechanism of target recognition by these antibodies and use this structural information to augment their potency. Finally, we will examine the therapeutic potential of these antibodies in a mouse model of lung cancer. The findings can lead to the discovery of new anti-tumor antibodies, revealing targets that may be superior in tumor binding and patient treatment over traditional existing ones.

Deciphering glioblastoma intra-tumoral heterogeneity

Non liver cancers rewire metabolism in the host liver

Submitted by Dr. Roei Mazor (Shulman and Sagi labs), Ran Salomon (Dahan lab) & Dr. Merav Shmueli (Merbl lab)

The goal of this study is to identify the optimal human IgG scaffold for therapeutic anti-PD-L1 Abs. To achieve this goal, we will mechanistically characterize the contribution of huFcgR pathways to the antitumor activity of FDA-approved PD-L1 Abs and will harness this new information to design optimal Fc variants as a framework for next-generation anti-PD-L1 Abs with improved activity.

Bone marrow (BM) recognition, homing and lodgment of long term repopulating hematopoietic stem cells (LT HSCs) are essential first steps for durable blood cell production during embryonic development and in clinical stem cell transplantation. Rare, BM retained LT HSCs endowed with the highest repopulation potential highly express EPCR (endothelial protein c receptor) and PAR1, which control LT HSC retention and chemotherapy resistance by limiting nitric oxide levels. We found that transplanted EPCR+ LT HSCs preferentially home to the BM, in contrast to immature progenitors. Intriguingly, EPCR+ LT HSCs can engraft the BM of non-irradiated recipients, while remaining quiescent, without giving rise to differentiated progeny. Strikingly, the quiescent homed EPCR+ LT HSCs were awakened by treating engrafted hosts with nitric oxide donor or with low dose chemotherapy, revealing that preconditioning and clearance of occupied HSC niches are not required.Importantly, we have treated human cord blood CD34+ stem and progenitor cells with a PAR1 mimicking peptide in vitro for 2 hours  which reduced their nitric oxide levels and doubled their homing to the bone marrow of transplanted immune deficient mice. Our study provides mechanistic insights concerning LT HSC homing, which may lead to improved BM transplantation protocols and be applied to prevent chemotherapy resistance of EPCR-expressing cancer stem cell