Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Table S1, Supplementary methods

Abstract The WRN helicase serves as a key target in the treatment of cancers characterized by microsatellite instability (MSI) because it plays a crucial role in resolving harmful non-canonical DNA structures that arise in cells with defective mismatch repair systems. Despite the critical functions of human DNA and RNA helicases, no drugs targeting these enzymes have been approved, largely due to the difficulties in identifying potent and selective inhibitors. In this study, we present the chemoproteomics-based identification of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This inhibitor specifically interacts with a cysteine residue (C727) within the helicase domain, which undergoes inter-domain movement during the DNA unwinding process. VVD-133214 reacts with the WRN protein in a nucleotide cooperative manner, promoting stable, compact structures that impede the enzyme's dynamic flexibility essential for its helicase activity. Inhibition of WRN by VVD-133214 leads to extensive double-stranded DNA breaks, nuclear enlargement, and ultimately cell death, specifically in MSI-high cells but not in microsatellite stable cells. The inhibitor demonstrated good tolerance in mice and significant tumor reduction in various MSI-high colorectal cancer cell lines and patient-derived xenograft models. Our findings highlight an allosteric strategy to inhibit WRN function that avoids interference from the endogenous ATP cofactor in cancer cells and positions VVD-133214 as a promising therapeutic candidate for patients with MSI-high cancers. Citation Format: Matthew P. Patricelli, Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodilles, Seth M .Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N Williams, Martha K. Pastuszka, John J. Sigler, Piergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Todd M. Kinsella. Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA003.

Abstract WRN helicase is a promising target for treating cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms. Currently, there are no approved drugs directly targeting human DNA or RNA helicases, in part due to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemical proteomic-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN. The clinical lead, VVD-214, covalently engages cysteine 727 of WRN in a nucleotide cooperative manner and inhibits ATP hydrolysis and helicase activity, resulting in widespread double-stranded DNA breaks, nuclear swelling, and cell death in MSI-high, but not microsatellite stable cells. VVD-214 provided robust tumor regression in multiple MSI-high colorectal cancer cell lines and patient derived xenograft models, including models derived from patients progressing on immune checkpoint therapies. VVD-214 was exceptionally well tolerated, and constitutes a promising oral drug candidate for patients with MSI-high cancers. Citation Format: Shota Kikuchi, Piergiorgio Pettazzoni, Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodiles, Seth M. Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N. Williams, Martha K. Pastuszka, John J. Sigler, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Matthew P. Patricelli, Todd M. Kinsella. Chemoproteomic-enabled discovery of VVD-214, a synthetic lethal allosteric inhibitor of WRN helicase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND11.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

PURPOSE:Cervical cancer is a viral-associated tumor caused by the infection with the human papilloma virus. Cervical cancer is an immunogenic cancer that expresses viral antigens. Despite being immunogenic, cervical cancer does not fully respond to immune checkpoint inhibitors (ICI). LIF is a crucial cytokine in embryo implantation, involved in maternal tolerance that acts as an immunomodulatory factor in cancer. LIF is expressed in cervical cancer and high levels of LIF is associated with poor prognosis in cervical cancer. EXPERIMENTAL DESIGN:We evaluated the impact of LIF on the immune response to ICI using primary plasmocytoid dendritic cells (pDC) and macrophage cultures, syngeneic animals and patient-derived models that recapitulate the human tumor microenvironment. RESULTS:We found that the viral proteins E6 and E7 induce the expression of LIF via the NFκB pathway. The secreted LIF can then repress type I interferon expressed in pDCs and CXCL9 expressed in tumor-associated macrophages. Blockade of LIF promotes the induction of type I interferon and CXCL9 inducing the tumor infiltration of CD8 T cells. This results in the sensitization of the tumor to ICI. Importantly, we observed that patients with cervical cancer expressing high levels of LIF tend to be resistant to ICI. CONCLUSIONS:Our data show that the HPV virus induces the expression of LIF to provide a selective advantage to the tumor cell by generating local immunosuppression via the repression of type I interferon and CXCL9. Combinatory treatment with blocking antibodies against LIF and ICI could be effective against cervical cancer expressing high levels of LIF.

Supplementary Data from Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis

Abstract T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2–7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.

Abstract The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K–mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E–mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti–PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. Significance: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

Cancer response to immunotherapy depends on the infiltration of CD8 + T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8 + T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8 + T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Abstract Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair. Experimental Design: Human HNSCC cell lines (n = 9), patient-derived xenografts (n = 9), tissue microarray (n = 194), TCGA expression data (n = 279), and primary tumor specimens (n = 10) were used to define the relationship between TGFβ competency, response to DNA damage, and type of DNA repair. Results: Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFβ signaling that increased response to radiation or cisplatin. TGFβ suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFβ signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFβ signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ). Conclusions: HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ’s role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy.

BACKGROUND:The transforming growth factor (TGF)-β pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-β pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-β isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-β2.MATERIALS AND METHODS:We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis.RESULTS:We observed that ISTH0047 is able to significantly reduce TGF-β2 mRNA and protein levels without altering the levels of TGF-β1 and TGF-β3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-β2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86.CONCLUSION:Overall, our data point to TGF-β2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

TGF-β signaling is commonly aberrantly activated in gliomas and other tumors and can exert a pro-oncogenic function. The authors identify a new mechanism for upregulation of TGF-β signaling in cancer. The deubiquitinase USP15 is shown to be able to bind the TGF-β receptor complex, counteract its degradation and potentiate its stimulation of downstream mediators. USP15 is amplified in human glioblastoma and could represent a therapeutic target, as its downregulation impairs the growth of glioblastoma cells in vivo .