Radisky, Evette S.

COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors—S/Ace2, Tmprss2, Cathepsins L and K, and Mpro—to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.

Abstract Tumor-associated fibroblasts (TAFs) exhibit an activated/fibrotic phenotype in all subtypes of non-small cell lung cancer. In contrast, we previously reported that lung TAFs exhibit premature senescence selectively in large cell neuroendocrine carcinoma of the lung (LCNEC), which is among the most aggressive subtypes of lung cancer. Moreover, we also reported that senescent fibroblasts enhance the growth and invasion of LCNEC cells in vitro and in vivo, and that the co-culture of LCNEC cells with normal fibroblasts was sufficient to induce fibroblast senescence. Intriguingly, whole-genome transcriptional profiling identified MMP1 as highly overexpressed in a panel of LCNEC cells versus non-LCNEC cell lines. Here we examined the role of MMP1 in LCNEC paracrine induction of fibroblast senescence. MMP-1 expression was silenced in LCNEC cancer cell lines by shRNA, and common senescent markers were analyzed after co-culture with normal fibroblasts, including β-galactosidase staining, cyclin-dependent kinase Inhibitor 2A expression (CDKN2A) and growth arrest. In addition, the tumor-promoting effects of fibroblast conditioned medium in LCNEC growth and invasion were measured. We confirmed that the LCNEC cell lines used in this study exhibited an increased expression of 3 neuroendocrine markers (CHGA, NCAM1 and SYP) compared to non-LCNEC cells. Induction of fibroblast senescence was confirmed after coculture with shScramble LCNEC cells. Moreover, knocking-down MMP1 in LCNEC cells was sufficient to abrogate fibroblast induced senescence upon co-culture, as well as the tumor-promoting traits of fibroblast's conditioned medium. The addition of active recombinant MMP1 (rMMP1) partially rescued the fibroblast senescent phenotype in co-culture with knocked-down MMP1 LCNEC cells, yet it was not sufficient to induce senescence when added to fibroblasts cultured alone. In contrast, treating fibroblasts with rMMP1 and the potent pro-fibrotic cytokine TGFβ1 was sufficient to induce both senescence and protumorigenic properties. Our results unveil a process of “niche construction” by LCNEC cells that is driven by their overexpression of MMP1, which induces senescence in adjacent fibroblasts that secrete factors that enhance the growth and invasion of LCNEC cells, thereby contributing to the aggressive nature of these tumors. In addition, our results reveal a new pathologic synergy between MMP1 and TGFβ1 in eliciting fibroblast senescence and enhancing its tumor-promoting traits. Moreover, our findings support that the aberrant carcinoma cell-fibroblast crosstalk mediated by MMP1 may be a suitable therapeutic target in LCNEC, which currently lacks targeted therapies. Citation Format: Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Evette Radisky, Noemí Reguard, Derek Radisky, Jordi Alcaraz. Large-cell neuroendocrine carcinoma cells of the lung induce a tumor-promoting senescent phenotype in fibroblasts through MMP1 overexpression and TGFβ1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5099.

We are presenting our on going studies with inhibitory research on Tmprss2, S-protein:Ace2, and 3CLpro using compound screening coupled with X-ray crystallography, molecular modeling, live virus screening using host human cells (BSL3 facility at UC Center for Infectious Disease and Vector Research, and organ-on-a-chip at Harvard Medical School for safety profiling before proceeding to animal models with InVivo BioSystems for ADMET. We have derived a useful chemical toolkit of 350 compounds for the community to study with biochemical assays and other biophysical-chemical studies that will prove useful in searching for optimal inhibitors of these targets to find suitable pharmacophores for blocking each of these enzyme's activities -- that would be beneficial for human health. Our past successes with these methodologies have resulted in over 28 patents, 11 technologies and two startup companies.

We are presenting our on going studies with inhibitory research on Tmprss2, S-protein:Ace2, and 3CLpro using compound screening coupled with X-ray crystallography, molecular modeling, live virus screening using host human cells (BSL3 facility at UC Center for Infectious Disease and Vector Research, and organ-on-a-chip at Harvard Medical School for safety profiling before proceeding to animal models with InVivo BioSystems for ADMET. We have derived a useful chemical toolkit of 350 compounds for the community to study with biochemical assays and other biophysical-chemical studies that will prove useful in searching for optimal inhibitors of these targets to find suitable pharmacophores for blocking each of these enzyme's activities -- that would be beneficial for human health. Our past successes with these methodologies have resulted in over 28 patents, 11 technologies and two startup companies.

Enzymes of the matrix metalloproteinase (MMP) family have been shown to play critical roles in cancer progression and metastasis, making them ideal targets for developing cancer therapeutics. MMP‐9 plays a significant role in tumor progression and metastasis of triple negative breast cancer (TNBC), while MMP‐3, the biological activator of MMP‐9, can also promote mammary carcinoma through inducing epithelial‐mesenchymal‐transition (EMT) in tumor cells. These enzymes are attractive as therapeutic targets; however, broad spectrum MMP inhibitors have proven ineffective, so inhibitors with greater selectivity are desired. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is a natural inhibitor of MMP‐3 and MMP‐9 with picomolar affinity, and offers a scaffold for engineering of selective MMP‐targeted therapeutics. To overcome the challenges of wide multispecificity of TIMP‐1 for different classes of MMPs, we used state‐of‐the‐art directed evolution and yeast surface display techniques recruiting high‐throughput library screening technology to fully evolve novel protein‐based drugs, based on the TIMP‐1 scaffold, with high selectivity for MMP‐3 or MMP‐9. We have developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized the top affinity and selectivity mutants with the catalytic domain of MMP‐3, and have solved the structure. These structures provide insights into the structural basis for improvements in binding affinity and selectivity. These studies and methodology developed in this project, will lay the foundation for developing novel therapeutic strategies for MMP‐related diseases and understanding protein‐protein interaction between MMPs and their inhibitors.

To facilitate investigations of protein–protein interactions (PPIs), we developed a novel platform for quantitative mapping of protein binding specificity landscapes, which combines the multi-target screening of a mutagenesis library into high- and low-affinity populations with sophisticated next-generation sequencing analysis. Importantly, this method generates accurate models to predict affinity and specificity values for any mutation within a protein complex, and requires only a few experimental binding affinity measurements using purified proteins for calibration. We demonstrated the utility of the approach by mapping quantitative landscapes for interactions between the N-terminal domain of the tissue inhibitor of metalloproteinase 2 (N-TIMP2) and three matrix metalloproteinases (MMPs) having homologous structures but different affinities (MMP-1, MMP-3, and MMP-14). The binding landscapes for N-TIMP2/MMP-1 and N-TIMP2/MMP-3 showed the PPIs to be almost fully optimized, with most single mutations giving a loss of affinity. In contrast, the non-optimized PPI for N-TIMP2/MMP-14 was reflected in a wide range of binding affinities, where single mutations exhibited a far more attenuated effect on the PPI. Our new platform reliably and comprehensively identified not only hot- and cold-spot residues, but also specificity-switch mutations that shape target affinity and specificity. Thus, our approach provides a methodology giving an unprecedentedly rich quantitative analysis of the binding specificity landscape, which will broaden the understanding of the mechanisms and evolutionary origins of specific PPIs and facilitate the rational design of specific inhibitors for structurally similar target proteins.

Abstract Ovarian cancer remains a challenging disease, with an average 5 year survival rate of 46%. At time of diagnosis, over 70% of patients have advanced metastatic disease, for which there are no effective treatment options. Of patients with metastatic disease, more than 70% of patients develop peritoneal carcinomatosis, making the peritoneum one of the main areas of dissemination. We have previously found that expression of the serine protease inhibitor Kazal type 1 (SPINK1) by ovarian cancer cells drives anoikis resistance, allowing the tumor cells to circumvent apoptosis protocols and facilitating tumor cell metastasis. For this new study we focused on ovarian clear cell carcinoma (OCCC), a histotype of ovarian cancer that has particularly poor prognosis if diagnosed in late stage. Using OCCC cell lines, we found that knockdown of SPINK1 reduces cell survival and stimulates apoptotic pathways in OCCC cells, when cultured under attachment-free conditions to mimic metastatic disease. We then identified Interleukin-6 (IL-6) as an upstream regulator of SPINK1 expression and a potential therapeutic target for metastatic OCCC: we showed that IL-6 silencing reduced SPINK1 mRNA expression and cell survival, while treatment with recombinant IL-6 increased SPINK1 expression and attachment-free survival. We developed a new OCCC mouse model and showed that knockdown of SPINK1 results in significant reduction of metastatic lesions, highlighting the impact of SPINK1 on OCCC metastasis. In ongoing studies we are testing intraperitoneal injections of FDA approved IL-6 inhibitors in a preclinical model to assess impact on abdominal dissemination of tumor cells. By targeting the IL-6 – SPINK1 signaling axis, we aim to develop therapeutic approaches to reduce the metastatic burden and peritoneal carcinomatosis of late stage disease, resulting in better prognosis and survival of patients with ovarian clear cell carcinoma. Citation Format: Christine Mehner, Erin Miller, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3758.

Abstract Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Intriguingly, senescent TAFs have been reported in a growing list of agressive cancer subtypes including large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in indirect co-cultures with transwells, revealing that paracrine signaling must be involved. A follow-up study identified MMP1 as an important regulatory protein in fibroblast senescence, since knocking-down MMP1 in LCC cells was sufficient to abrogate the induction of fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. Here we examined the potential role of the protease activated protein 1 (PAR-1) in fibroblast senescence by MMP1, since PAR-1 can be activated by MMP1 and has been reported in the stroma of lung cancer patients. We found that PAR-1 expression was down-regulated in fibroblasts that become senescent upon co-culture with LCC cells. In addition, forcing a reduced PAR-1 expression in fibroblasts by siRNA increased their expression of markers of the senescence associated secretory phenotype (SASP) during co-culture with LCC cells, whereas the percentage of senescence-associated beta-galactosidase positive fibroblasts remained largely unaffected. These results suggest that fibroblast senescence elicited by MMP1 secreted by LCC cells is mediated by processes other than PAR-1 activation and/or overexpression in fibroblasts. Citation Format: Marta Gabasa, Rafael Ikemori, Evette Radisky, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2021.

Abstract Large cell carcinoma (LCC) is among the most aggressive histologic subtypes of non-small cell lung cancer, but the mechanisms underlying such aggressive nature remain unknown. We recently showed that fibroblasts from LCC patients exhibit premature senescence in vitro, and that co-culturing LCC cells (but not cancer cells from other lung cancer subtypes) with normal fibroblasts in transwells is sufficient to induce senescence in the latter in an oxidative stress-dependent manner, supporting that fibroblast senescence is induced by a secreted factor(s) from LCC cells. Remarkably, we also found that senescent fibroblasts secrete factors that stimulate the growth and invasion of LCC cells beyond the stimulation elicited by nonsenescent fibroblasts, revealing that fibroblast senescence may contribute to the aggressive nature of LCC. Whole-genome transcriptional profiling of a panel of lung cancer cell lines identified MMP1 among the genes with larger expression in LCC compared to other lung cancer subtypes. Since MMPs can induce oxidative stress, we examined whether the excessive MMP1 expression in LCC cells was involved in the induction of fibroblast senescence in co-cultures. Knocking down MMP1 in LCC cells was sufficient to abrogate fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. In contrast, autophagy, which has been previously associated with fibroblast senescence in breast cancer, was not upregulated in fibroblasts upon co-culture with LCC cells. These results support that the selective aberrant expression of MMP1 in LCC cells plays a major role in their ability to induce a protumorigenic senescent phenotype in adjacent fibroblasts through a mechanism that is independent of autophagy. Moreover, our observations identify MMP1 as a potential therapeutic target against the aberrant cancer cell-fibroblast crosstalk in LCC. Citation Format: Jordi Alcaraz, Marta Gabasa, Rafael Ikemori, Noemí Reguart, Evette Radisky, Derek Radisky. Cancer cells induce a protumorigenic senescent phenotype in fibroblasts through MMP1 but not autophagy in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 994.