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The clinical approvals of KRAS G12C inhibitors have been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor’s KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters α and λ. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final “K20” model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV’s TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown clinical promise, there is still a need for therapies that overcome resistance and target non-KRASG12C mutations. KRAS activates downstream MYC, which is also a difficult-to-drug oncoprotein. We have developed an \"inverted\" RNAi molecule with the passenger strand of a MYC-targeting siRNA fused to the guide strand of a KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5'-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a more than 10- to 40-fold improvement in inhibition of cancer viability in vitro. When conjugated to an EGFR-targeting ligand, the chimeric siRNA was delivered to and internalized by tumor cells. As compared with individual targeting siRNAs, the chimeric design resulted in considerably improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in multiple cancer models. This inverted chimeric design establishes proof of concept for ligand-directed, dual silencing of KRAS and MYC in cancer and constitutes an innovative molecular strategy for cotargeting any two genes of interest, which has broad implications.

Activating mutations in the GTPase KRAS are responsible for driving nearly 25% of all cancer types. Despite extensive research, effective KRAS inhibitors remained elusive until recently. The discovery of covalent molecules that irreversibly bind to the KRASG12C mutant protein, present in approximately 15% of cancer patients, facilitated the development of KRASG12C-specific inhibitors. However, clinical evidence of rapidly acquired resistance to KRASG12C inhibitors underscores the need for therapies that can overcome these challenges and target non-KRASG12C mutations. Mutant KRAS cooperates with the transcription factor MYC to promote tumorigenesis, and dual targeting of KRAS and MYC has shown synergistic anti-cancer effects in murine models. We have developed highly potent siRNAs against KRAS and MYC that inhibit downstream signaling and key cancer phenotypes. Using an endo-nucleolytic DNA bridge, we created an RNAi molecule that link MYC- and KRAS-targeting siRNAs in “serial” and “inverted” chimeric conformations. Compared to both serial chimeras and individual siRNAs, the inverted chimeric siRNA designs achieved superior gene silencing, resulting in ~40-fold reduction in cell viability and loss of tumorigenic potential. Mechanistic studies reveal that endosomal cleavage of the chimeric siRNA generates 2’-deoxythymidine (dT) 5’-terminal overhangs on the antisense strand, further enhancing target silencing in a dose-dependent manner. This chimeric siRNA design establishes proof-of-concept for dual KRAS and MYC silencing and represents a new molecular strategy for co-targeting any two genes of interest with broad implications.

Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown clinical promise, there is still a need for therapies that overcome resistance and target non-KRASG12C mutations. KRAS activates downstream MYC, which is also a challenging-to-drug oncoprotein. We have developed an \"inverted\" RNAi molecule with the passenger strand of a MYC-targeting siRNA fused to the guide strand of a KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5'-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a >10-40-fold improvement in inhibiting cancer viability in vitro. When conjugated to an epidermal growth factor receptor (EGFR)-targeting ligand, the chimeric siRNA was delivered to and internalized by tumor cells. As compared with individual targeting siRNAs, the chimeric design resulted in considerably improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in multiple cancer models. This inverted chimeric design establishes proof-of-concept for ligand-directed, dual-silencing of KRAS and MYC in cancer and constitutes an innovative molecular strategy for co-targeting any two genes of interest, which has broad implications.Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown clinical promise, there is still a need for therapies that overcome resistance and target non-KRASG12C mutations. KRAS activates downstream MYC, which is also a challenging-to-drug oncoprotein. We have developed an \"inverted\" RNAi molecule with the passenger strand of a MYC-targeting siRNA fused to the guide strand of a KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5'-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a >10-40-fold improvement in inhibiting cancer viability in vitro. When conjugated to an epidermal growth factor receptor (EGFR)-targeting ligand, the chimeric siRNA was delivered to and internalized by tumor cells. As compared with individual targeting siRNAs, the chimeric design resulted in considerably improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in multiple cancer models. This inverted chimeric design establishes proof-of-concept for ligand-directed, dual-silencing of KRAS and MYC in cancer and constitutes an innovative molecular strategy for co-targeting any two genes of interest, which has broad implications.

The clinical approvals of KRAS G12C inhibitors has been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor’s KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final “K20” model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multi-modular scaffold, GIV (a.k.a Girdin) titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (LPS, a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress pro-inflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene depletion studies confirmed that the presence of GIV ameliorates DSS-induced colitis and sepsis-induced death. The anti-inflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL)-motif which binds the cytoplasmic TIR-modules of TLR4 in a manner that precludes receptor dimerization; the latter is a pre-requisite for pro-inflammatory signaling. Binding of the TILL motif in GIV to other TIR modules inhibits pro-inflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses. Competing Interest Statement The authors have declared no competing interest.