R32 Catalyst Application Review Submissions; CRB

This is a collaboration between two tenured faculty members, McNerney, an expert on CUX1, and Yue, director of the Center for Cancer Genomics. Somewhat unclear from the proposal which assays require Yue's expertise or if they are pushing the limits of established technology.

This proposal seems to me like a natural next step for McNerney's successful research program. Results would undoubtedly improve our understanding of CUX1. The question for me is whether the Catalyst mechanism is needed for this work or could it be funded by the NIH as-is?

The proposal is logically structured and the experimental plan seems solid.

I find the problem this team is going after to be appealing, and the approaches range from biophysical (microscopy) to quantitative biochemistry. I did find the proposed plan to not indicate a tight collaboration. It is nearly like one lab will develop one Aim (TI-C), and the other lab will develop the other (oligoPAINT of the hox locus). It's not clear how much progress in one will help with the other.

Both parts of this proposal seem technically quite challenging. It would certainly be an impressive achievement to obtain quantitative, single-cell distributions of genome structure, and in that sense it is high reward.

I wish the proposal had elaborated more on expected or possible outcomes. What is the best case outcome here in terms of discovery? Or is the goal chiefly assay development?

This is an exciting collaboration that spans quite different areas of biology and chemistry and is a good senior-junior pairing.

I find this proposal quite exciting in the sense that they may "observe a lot just by looking" (to paraphrase Yogi Berra). It's very possible that this will lead directly to the discovery or isolation of an important nausea-inducing factor in serum.

There are many reasons that this might not work--the active circulating factors might not be proteins or peptides, they might be active at very low concentrations, etc. Still it seems that the chances of success are good. Further, this project has an appealing "fail fast" quality. Presumably a finite list (maybe with of length zero!) will come out of the mass spec comparison between motion sickness and normal conditions, and these can be systematically evaluated.

Investigators are highly qualified. There is history of collaboration.

In specific aim 1, the investigators will define and model the nutrient microenvironment in breast tumors. First, quantitative metabolomics of tumor interstitial fluid will be utilized to define the TNBC nutrient microenvironment. Next, the investigators will develop a custom TNBC culture model that replicates the TNBC nutrient microenvironment. In specific aim 2, the investigators will profile the impact of the nutrient microenvironment on response to therapeutic agents via a drug screen platform. In addition, the nutrients, and alterations in cellular processes responsible for differential drug responses will be identified.

Overall, the level of innovation is high. However, there are a number of important caveats. If successful, the study may yield results that will advance the treatment of patients with TNBC.

The investigators are highly qualified. The environment is suitable for the proposed studies, and they will be able to gain access to biospecimens.

Aim 1, will establish correlations between ER Ca superscript 2 + ER Ca storage and CAR T cell thickness. Experiments are proposed to determine the relationship between ER Ca storage and immunosuppressive culture conditions of T cells. The experiments will be repeated with primary T cells from healthy and cancer patients. In specific aim 2, a correlation between ER Ca stores as a function of days after cell infusion and patient survival will be performed. This will utilize patient’s biospecimens from patients who are undergoing CAR T therapy at Northwestern University, where Dr Lin is a cellular therapist.

The proposal focuses on evaluation of T cell thickness as a strategy to understand the potential to predict CAR T therapy outcomes. The objective of the application is to develop a cost-effective imaging technology for quantitative evaluation of T cell health. The hypothesis is that the endoplasmic reticulum (ER) calcium storage correlates with T cell function status and to clinical response of CAR T therapy.

The investigators are highly qualified to complete the research proposal.

The focus of the application is on non-small cell lung cancer and the investigators propose a platform for in vitro modeling of non-small cell lung cancer. Specific aim1, is focused on demonstrating and optimizing the microfluidic platform for position 3D models of non-small cell lung cancer. The investigators have developed a spheroid culture Micro Wells that will be used to develop and optimize the device for cell lines that have KRAS mutations will be co-cultured with CAFs and with PBMCs. The microfluidic system appears to be in place and will be used in specific aim 2, to test drug efficacy. The drugs to be tested are not known. Single cell transcriptomic analysis of the patient derived organoids will be performed.

Overall, the proposal is likely to be successful. However, the use of 3D cultures and patient derived organoids mixed with PBMCs is not innovative. This approach has been tested in multiple previous studies. It is also not clear how histocompatibility of PBMCs that will be mixed with the cell lines will not lead to issues with compatibility.

This collaborative team consists of a relatively new investigator (Lee; Assistant Professor) from Northwestern and a more established investigator (Moellering; Associate Professor) from UIC. The expertise of both investigators is strong and complementary. Lee is an expert in the neurobiology of taste and chemosensation. Moellering is an expert in chemistry, proteomics and metabolomics. The potential synergy between the investigators to answer the proposed questions is very strong.

This is an extremely compelling, competitive, and high-risk/high-reward project. The neurobiological and chemosensory basis for nausea is not only an emerging and fascinating area of study, but highly relevant to health and disease. Current treatments for nausea and motion sickness are primitive at best. The discovery of the long-hypothesized systemic chemicals that promote nausea would be a groundbreaking event, and could potentially revolutionize our understand and treatment of the distress it causes.

The proposed experiments are innovative and feasible based on the expertise of the investigators and on the preliminary data. Dr. Lee has been actively working on neurons that trigger the perception of nausea in the brain. Preliminary data show that they can identify neurons that are activated by nausea, and they can culture these cells for functional assays. The latter is very important and novel. Dr. Moellering has expertise in proteomics and metabolomics. Their preliminary microproteomics data indicate that they can identify novel compounds in CSF of nauseated mice. The challenge here will be to wade through the potentially large number of chemicals that are differentially found in nauseated animals. Presumably, the bioassay (in vitro imaging) will allow them to screen individual hits. This is a powerful plan with substantial but reasonable risk.

A proposed 3D microfluidic platform may be highly significant in that it addresses a need in cancer diagnostics for approaches that recapitulate the tumor microenvironment (TME) ex vivo using small quantities of patient-derived samples. Specifically, the proposed device would replicate the TME by facilitating the growth of patient-derived organoids (PDOs) of defined size with control of composition (e.g, cancer-associated fibroblasts (CAFs), peripheral blood mononuclear cells (PBMCs), and extracellular matrix (ECM)). Moreover, the microfluidic platform would enable controlled addition of drugs, manipulation of physical parameters and downstream analyses of TME effects on cell-cell communication, genetics and development of drug-resistance. One minor weakness of the proposal is that the proposed device is not compared against the state-of-the-art in terms of microfluidics as applied to in vitro TME models. Have other similar approaches been reported and how would they compare to the proposed approach?

Approach will demonstrate feasibility of 3D microfluidic platform: Aim 1, build and test device based on published spheroid microwells, grow PDO organoids and assess phenotypes; Aim 2, Quantitatively evaluate drug resistance in TME with statistical significance. Criteria for success are rigorously defined including culture growth curves and phenotypes, drug resistance profiles and throughput of experiments. No weaknesses.

PI team is highly complementary. Shimanura and Papautsky (UIC) have established collaboration with publication record. Basu (UC) brings inter-institutional collaboration in form of needed genetic analysis. A clear plan for seeking funding is delineated. No weaknesses.

Proposed -omics study of mouse cerebral spinal fluid (CSF) addresses lack of knowledge of the underlying molecular basis of nausea. Nausea is a widespread and costly side-effect of various underlying conditions and medications, and its molecular basis is poorly understood. Current understanding is that chemoreceptors detect circulating toxins, pathogens and other alarm signals produced in the body to initiate nausea; the molecular identities of these circulating factors are unknown. The project would identify altered proteins and metabolites in mouse cerebral spinal fluid (CSF) using established methods following induction of nausea under controlled conditions using a novel motion sickness (MS) induction strategy. Significance is potentially high, as identification of nausea-inducing molecules could generate new hypotheses and research directions.

PIs Lee (NU) and Moellering (UC) bring complementary expertise in the model system and omics capabilities respectively. They have already initiated studies on the project, creating a motion sickness method of nausea induction, generating preliminary proteomics data, and establishing that motion sickness increases neuronal activity in the purported nausea chemoreceptor zone. Catalyst funding would enable more expansive research.

Published and preliminary results indicate that the approach is likely to be successful: i) the model system can induce nausea in mice in a controlled and reproducible way; ii) the proteomic and metabolomic capabilities are well established; iii) and calcium imaging would yield data of effects of identified factors on neuronal signaling. The high likelihood of success is positive. There is some risk that the data generated will be of limited value, but the underlying rationale for the proposed studies is strong.

Very good inter-institutional collaboration between two assistant professors

The question being addressed is very important

It may be that ER calcium levels will be a good measure of T cell fitness and correlate with the effectiveness of CAR T therapy, but if that is not the case little will be learned from this study.

Both investigators as well as the collaborator at U. Rochester are well positioned to do the studies. Their expertise complement well.

1. This proposal falls under high-risk High-reward category. The work will attempt to model the metabolic micro-environment in triple negative breast tumors in order to assess if nutrient micro-environment play a role in differences seen in therapeutic efficacy of cancer drugs. The approaches being taken are highly challenging and therefore accurately mapping/identifying metabolites in tumor micro-environment will be challenging.

The results from such modeling studies in genetically identical mouse models or PDX models may not mimic what is seen in genetically heterogenous human population. Genetic polymorphisms are known to be a major variable in drug sensitivity, which is not mentioned or being considered. Although metabolic micro-environment plays a role in variations in drug sensitivity/ efficacy other more prominent factors such as immune-modulatory cytokines and immune cells within the tumor micro-environment play a larger role in dampening the efficacy of chemotherapy and other drugs, which is not considered.

Inter-institutional collaboration is strong and complementary. The two investigators presently collaborate on projects with the same theme and has a manuscript under revision. It is unclear and not mentioned what experiments will be carried out by Dr. Horvath at NW other than mentioning his interest in IFN and acting as a mentor. It seems like almost all the work discussed and proposed will be carried out by Mendoza lab.

The proposal is conceptually innovative and falls under high-risk, high-reward category. The concept of being able to modulate signal intensity of JAK/STAT pathway based on receptor orientation and engineering or identifying drugs for this purpose is innovative and falls under high reward category. The approaches taken to test their concept is less innovative and or risky.

It is unclear and not mentioned what experiments will be carried out by Dr. Horvath at NW other than mentioning his interest in IFN. It seems almost all the work discussed are from Mendoza lab. The proposed concept if proven especially beyond the IFN field will have a major impact on new drug development and selective treatments for many clinical conditions that are know to involve JAK/STAT pathway and their cognate receptors. There are no concerns on the feasibility.

The project if successful will have a great clinical impact and create a path for developing new class of drugs that work as modulators as opposed to inhibitors or activators.

The collaboration is highly appropriate. Dr. Rosner brings well established expertise in signaling pathways and networks and Dr. Pauli brings expertise in natural product isolation and characterization.

The proposed study present moderate risk and moderate reward.

1. Innovation of the proposed cannabis-interferon relationship. 2. Risk reward relationship well suited for this award mechanism. 3. Mechanism based approach of Aim 2. 4. Interdisciplinary team with strong backgrounds in natural product isolation and characterization (Pauli) and signaling pathways and networks (Rosner). Moderate Weaknesses: 1. Modest level of activity of CBD in the SARS-CoV-2 replication assay (IC50 ~1 uM). 2. Modest significance for translation (e.g. is this a good lead candidate for chemical optimization or alternatively what are the chances that other cannabinoids will be significantly more potent). 3. Induction of interferon by small molecules is already an intensively studied area. Minor Weakness: The team would benefit from additional expertise in virology.

The strengths and the weaknesses balance out and the score is driven by the observation that many others have worked on developing the induction of interferon by small molecules and that this work while interesting is of moderate potential impact.

Appropriate collaboration between two junior investigators with complementary expertise (Dr Hu in immune imaging and Dr. Lin clinical cellular treatments of lymphoma).

The proposed studies are high-risk because they are based on the hypothesis that T cell fitness drives patient response to CAR T cell therapies and high-reward due to their potential to improve CAR T cell treatment efficiency

Strengths: 1. The significance of efforts to improve CAR T treatment efficacies in a rapid and cost efficient manner. 2. Potential to improve CAR T treatment efficacies by surveying T cell fitness. 3. Innovation of the study to determine T cell fitness by Ca2+ imaging. 4. Preliminary data supporting the feasibility of the study. 5. Consideration of pitfalls and alternative strategies. 6. Access to patient pools with consideration of statistical power. 7. Appropriateness of this award mechanism for 2 junior PIs, which are rising stars. Weaknesses: 1. A minor weakness is that the long-term impact is based on the hypothesis (to be tested) that T cell fitness is the most significant reason for poor outcomes in T cell therapy.

The strengths of this proposal greatly outweigh the weaknesses, particularly the potential relevance to human health.

strong collaboration between groups with complementary expertise. Some previous collaboration, but this is a new project

Using state of the art approaches to study an important basic science question

DNA barcoded antibodies was not explained or referenced

The pending proposal "Mapping protein signatures to single allele chromatin topologies at genomic resolution" would seem to have substantial overlap with the present proposal

1. Relevant expertise is balanced equally between Dr. McNerney, who is a leading expert on CUX1 and Dr. Yue, an expert in 3D genome architecture. I wish there was more information on their prior interactions and planned future collaboration, but the project seems to be a good pairing.

2. The project fundamentally addresses a basic scientific question, looking at the role CUX1 plays in genome architecture and DNA looping, which is critical in gene regulation. But it also could directly address clinical observations where CUX1 mutations are associated with myeloid neoplasia, and haploinsufficencies are associated with hematopoiesis and cancer. Therefore, findings could help explain mechanisms of disease.

3. The proposal makes use of latest technologies, including HiC mapping and Degron targeting. These will be used to generate new cell lines and acquire datasets that are novel.

4. The proposal is focused with a strong attention to detail and specific descriptions of outcomes. The work will generate large datasets, which itself has strengths and weaknesses; positively, this will serve as a basis for external funding, but negatively, it is unclear how all data will be interpreted.

This team is comprised of Dr. Horvath who is a full professor at Northwestern with more that 30 years experience in studies of interferon signaling in response to viruses. He is partnering with an Assistant professor, Dr. Mednoza, who has been at UC since 2018. The pair have complementary expertise that is a major positive for this award. The inclusion of an assistant professor and particularly the description of the mentoring relationship between Dr. Horvath and Dr. Mendoza is a strength as aligns well with the CBC extra considerations

This application is particularly high risk, high reward in nature. There is preliminary to support the concept and technical approach for both aims, but the hypothesis under consideration are particularly forward thinking. The risk of course of all HTP screens is that nothing will be found or that what is found will not be valuable. The project has built in secondary screens and Dr. Horvath is the head of the NU HTP lab so there is strong support that the experiment is well designed. Aim 2 has worked for one receptor-kinase pair, and well test if the concept expands to other pairs. It is a worthy hypothesis. The high risk nature of this work is a merit for the catalyst mechanism.

Strength This work is well grounded in the preliminary finding that affinity can impact signaling. The plan is to test if this aspect of signaling can be leveraged for discovery of host directed antiviral compounds. Aim 1 is well designed with multiple validation steps and a plan to pursue hits in the HTP screen with appropriate validation The design of Aim 2 is based on cool preliminary data that used structure bases concepts to design optimized receptor kinases that are tuned. The experiment will test if the concept will be expanded. Findings in Aim 2 could have impact across many different signaling systems. Weakness Perhaps it is the very short format and the details for the structure variation screen are quite nuanced, but was not exactly clear from the method how the validation of different signaling amplitudes in chimeras would be validated in the full length proteins.

This is a team of two assistant professors with complementary expertise to address a new research direction for both investigators. Dr. Muir started at UC in 2019 and has expertise in metabolism in the context of tumor cells. This group can measure nutrient availability in tumors. Dr. Coloff is an Assistant professor at UIC since 2018 with expertise in breast cancers using 3 D organoid models. The two will work together to leverage Dr. Coloff's experience in TBNC with Dr. Muir in metabolomics of cancer to pursue questions of TME in breast Cancer. The alignment of this strong pair of assistant professors to pursue a new research direction for both groups is a strong alignment to the extra consideration criteria for this application

This work stands on solid ground for prior studies using pancreatic cancer but will be a new direction for Breast cancer. The screening aspect of Aim 1 makes this work high risk and not likely to be funded. However, if interesting trends are discovered, the work is likely to generate hypothesis that will be fundable with potential for a deeper understanding of chemotherapeutic strategies to target TNBC, which has low success rates of treatment and needs advanced research. The high risk aspect of Aim 1 and Aim 2 is a positive for this award.

Strength The study is well within the expertise of the collaborators and thus likely to give results that can be meaningful interpreted. The study leverages technical expertise previously applied to PDAC to another cancer that also has poor outcomes The plan to develop a culture model that will better replicate the TME of TNBC has strong potential for testing of currently used drugs (Aim 2) but also for testing of novel compounds in the future. The plan to use mice although not optimal, is well justified and the investigators are correct that other options are not viable with being harmful to patients. Weaknesses The investigators are open about a weakness of not being able to test all drug in use, but there seems to be many that can be tested.

Excellent collaboration between two groups with complementary expertise

Important question addressed using state of the art approaches

May be well enough developed for national funding

This is a strong collaborative team each with appropriate expertise to the collaboration. The team has been working together since 2020 on a de novo project to demonstrate how CBD impacts SARS-CoV-2. Dr. Pauli is a full professor at UIC with natural products expertise. He will manage Aim 1 to improve purification of CBD molecules and to source CBD molecules. Dr. Rosner is a full professor at UC with long term record in cell signaling, in particular related to Map kinases, UPR, and IFNs. This is a new direction of research for both investigators. They have joint paper in Science Advances on this project in 2022. Weakness: The investigator team does not meet any of the extra consideration categories. The lack of junior investigator tempered my score for the team.

This project may have been high risk at its conception in 2020 and has led to a significant paper in 2020. However, the plan as put forward now is to conduct a more detailed analysis of how CBD activate innate responses through NRF2. This as written is a very exciting project that is likely to be successful, but I would not consider it high-risk. The lack of a high risk component tempered my score for an otherwise outstanding proposal.

The project is highly feasible and likely to succeed. Strengths The preliminary data support that CBD inhibits SARS-CoV-2 replication although other CBD-like compounds are less effective. There is much enthusiasm of testing these related compounds for SARS-CoV-2 infection in cell lines (Aim 3). The proposed model of CBD activation of ISGs is testable with defined strategies in expertise of the investigator. Weakness The signaling responses (proteins arrays etc) in Aim 2 seem to be intended to be performed for CBD impact on cells. No plan to monitor signaling in response to SARS-CoV-2 or how the signaling response to SARS-CoV-2 in A549 cells will differ in the context of CBD. There is a plan to test replication of virus in KO cell lines, but this is less interesting than if CBD alters the signal flow. This is particularly important as SARS-CoV-2 has many strategies itself to suppress IFNs.

Team collaboration is exceptional. The questions being addressed match each PI's expertise in a synergistic fashion. Ruthernburg lab has world class expertise in mapping 3D chromatin structure and Spille lab has done pioneering work on super resolution microscopy. Their team efforts are complementary and will address the aims of the proposal employing cutting-edge technology to develop new platform/assay for answering basic science questions with great translational impact. The two investigators have collaborated on completely different project in the context of a student Ph.D. thesis.

This is a true high-risk, high-reward project. Both investigators have the foundation to take on the questions being addressed. There are two aspects to the project. The investigators plan to push the MicroC technology currently being used to decipher short and long range changes in chromatin looping and chromosomal interactions from a qualitative assay to a quantitative one. In addition they will attempt to answer a long-standing question with regards to if 3D structural changes precedes epigenetic modifications such as histone modification and DNA methylation or the reverse is true. Mapping such temporal events leading to gene transcriptional changes focused on 3D chromatin structure is very high risk with very high rewards in terms of both discovery science and translational potential.

The work will attempt to answer whether structural changes in chromatin architecture precedes epigenetic chromatin modifications or epigenetic modifications are necessary in order for structural changes in chromatin to occur. The work will pave the path for the chromatin conformation capture technique to be quantitative in an absolute scale. The integration of two cutting edge technologies ie oligoPaint chromatin tracing approach and DNA bar-coded antibodies against histone marks to decipher whether histone marks on chromatin precedes chromosomal structural change is quite ambitious and unique. This approach has been utilized in other contexts and therefore is quite feasible. Both investigators have a very strong track record in their respective fields (Chromatin architecture determination and super-resolution microscopy) and therefore has an excellent chance in succeeding in tackling the questions being addressed. The use of Hoxb cluster activation system in mouse ESCs as their model is an excellent choice. No major weaknesses are noted

Developing chromatin conformation capture technique as a quantitative assay will be quite powerful in uncovering transcriptional changes due to extracellular cues in a dynamic fashion.

The applicants have shown that cannabidiol (CBD), the dominant ingredient in hemp, inhibits SARS-CoV-2 replication in cultured cells and mice. This collaborative proposal pairs two PIs with very different, and complementary, expertise.

Pauli and the UIC group have spearhead enabling technologies to support interdisciplinary drug discovery from natural products. The first aim will source high quality cannabinoids and improve methodology for their purification and quality control. Hemp cannabinoids are largely unregulated and contain > 300 other cannabinoids in addition to CBD. Please note that I have no expertise on this topic. My evaluation is based strictly on my reading of the proposal (which I found very interesting).

In Aim 2 the Rosner group will identify the mechanism by which CBD activates anti-viral interferon signaling and then (in Aim 3) examine whether any of the other cannabinoids identified in Aim 1 are more effective at inducing interferons and blocking SARS-CoV2 infection.

The criteria for measuring success seem achievable. The questions being addressed are important and timely.

Very well written proposal combines the complementary expertise of a senior and junior investigator.

A similar proposal submitted to Abbvie Ventures was a semi-finalist.

There's a risk that they won't identify small molecule modulators but a high chance of translatable reward that could lead to novel and impactful new drug development

The investigators are excellent. Papautsky directs CADMIM which is a NSF supported Industry-Academic consortium, is on the Lab on a Chip Advisory board, and has pioneered many microfluidic innovations. Shimamura is a cancer researcher and has previously published an aspect of this proposal with Papautsky. Basu developed dop-seq and brings single cell transcriptomics expertise to study TME.

The project involves developing a simple microfluidic trap array that can be scaled to clinically relevant samples. This approach would allow rapid screening of PDOs which is currently labor intensive. Their aims will provide preliminary data to pursue other NIH proposals. If successful it could improve patient outcomes.

Their research plan is well thought out. They have good criteria for success that will compare their results to gold standards which take 3-4 weeks as compared to 5 days of theirs. The device is conceptually very feasible and while they have not made this before, should not be too difficult to fabricate.