GDP-bound State Research Articles

Conformational Dynamics Allows Sampling of an “Active-like” State by Oncogenic K-Ras-GDP

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the “active-like” conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

Abstract 2975: RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC

Abstract Background: RAS is the most commonly mutated oncogene in cancer, with KRAS mutated in ~30% of non-small cell lung cancer (NSCLC). RAS is a small GTPase cycling between GTP-bound ‘ON’ state and GDP-bound ‘OFF’ state. KRAS oncoproteins cycle between these states via hydrolysis and nucleotide exchange with the aid of GAPs, including NF1, and GEFs. Given the ’inactive state’ inhibition of recently developed KRAS G12C inhibitors on the GDP-bound state, this has highlighted the continued reliance of KRAS-mutants upon cycling, and the potential for mutants to retain dependence upon upstream influences, including NF1, for pathogenicity. Methods: 474 patients with advanced RAS- and/or NF1-mutant NSCLC were retrospectively identified from four tertiary cancer centers between 2008 -2021. DNA from archival FFPE samples, serum or combination underwent targeted NGS panels to identify mutations. Molecular, clinical, pathological and treatment outcome data were collected. Online resources including cBioPortal and Project Achilles were used to assess the functional role of any findings. Results: KRAS mutations were identified in 416/474 patients and NF1-mutations in 63/474 patients, eight of whom harbored two NF1-mutations. 24/63 (38%) of NF1-mutant cancers had a concomitant KRAS-mutation. We identified that KRAS G13D was more prevalent in NF1 mutant cancers vs. NF1 wildtype (NF1 MT: 6/24, 25%; vs. NF1 WT: 4/281, 1.4%; p<0.0001). KRAS G12C was identified in 11/24 (45.8%) of the double mutants vs. 109/282 (38.7%) of the NF1 WT patients (p=0.52). Those with G13D/NF1 co-mutation had a predicted pathogenic NF1-mutation in 5/6 (83%) of cases. Functional analysis of NF1 KO in G13D mutant lung cancer cell lines identified that NF1 was more essential in G13D mutant cell lines, with median Chronos score -0.26 G13D vs. -0.04 G12C (p=0.02). mRNA expression data identified a range of mRNA expression Z-scores relative to all samples for NF1-mutant cancers, ranging from lowest expression at -4.54 to +1.63, median -0.51. A higher mRNA expression was identified for NF1 missense mutations of unknown significance compared to truncating mutations with likely pathogenic, loss of function. Conclusions: These results highlight the co-mutational landscape of KRAS G13D with NF1 in NSCLC, suggesting functional importance conferred by NF1 loss, and highlight NF1 mutations as an additional pathogenic even when combined with KRAS mutation. The genomic landscape of KRAS and NF1 mutant NSCLC will be explored further through Whole Genome Sequencing data from the 100,000 Genomes Project (Genomics England). Citation Format: Helen M. Adderley, Mihaela Aldea, Jaqueline Aredo, Mathew Carter, Matthew Church, Pantelis Nicola, Jamie Weaver, Aisha Ghaus, Damien Vasseur, Matthew Krebs, Nicola Steele, Fiona Blackhall, Heather Wakelee, Benjamin Besse, Colin Lindsay. RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2975.

Abstract 2918: Comprehensive survey of KRas4B membrane interaction and orientation

Abstract Three RAS genes encode four Ras proteins, HRas, NRas, and KRas splice variants, KRas4A and KRas4B. In human cancers, KRAS oncogene is highly expressed (85%) as compared to other RAS oncogenes, NRAS (11%) and HRAS (4%). Oncogenic Ras leads to aberrant cell proliferation signaling, primarily through the MAPK (Raf/MEK/ERK) pathway. At the plasma membrane, nanoclustered Ras proteins promote Raf activation, mediating the downstream signaling pathway. Among the Ras isoforms, KRas4B in the membrane has been studied extensively due to its higher level of mutations in cancer. Although a number of experimental studies have focused on KRas4B subcellular localization and their nanocluster formation at the signaling platform, the mechanistic details of the membrane anchorage and the membrane orientation of KRas4B at atomic resolution are still unknown. Here, comprehensive computational studies were performed for KRas4B at the anionic lipid bilayers, composed of the phospholipids, PS and PA, and the phosphoinositides, PIP2, PI4P, and PI5P. Wild-type and two oncogenic mutants, G12D and G12V KRas4B in the GTP- and GDP-bound states were considered. The lysine rich HVR was post-translationally modified with the farnesyl and methyl groups. Previous studies showed that wild-type KRas4B-GDP interacts weakly and nonspecifically with anionic phospholipids, while oncogenic mutations alter selective anionic lipid binding toward anionic lipids, PA, PI3P, PI4P, and PI5P. Our studies indicate that the membrane interaction of KRas4B is highly sensitive depending on the types of nucleotides, mutational states, and anionic lipids enriched the lipid bilayer. While the GTP-bound spontaneously inserts its prenyl group into the hydrophobic core of the bilayer, the occurrence of spontaneous insertion is reduced in the GDP-bound form. The distance of GTP-bound catalytic domain from the bilayer surface is longer than the GDP-bound form. The HVR is sequestered by the GDP-bound catalytic domain, which causes the catalytic domain to be occluded and increases the membrane interaction by the catalytic domain. In contrast, the GTP-bound catalytic domain releases its HVR and liberates from the membrane interaction. The occlusion of catalytic domain is less prevalent in the GTP-bound state. KRas4BG12D/G12V-GTP exhibits the active-state orientation, exposing its effector binding site for recruiting the effectors. While PA and PIP2 support the active-state orientation, the PIP4 enriched bilayer prevents the spontaneous prenyl insertion resulting in the occlusion of catalytic domain. Our results underscore the importance of detailed structural mechanisms of KRas4B membrane interaction and orientation, elucidating vital mechanistic role of KRas4B, a key membrane-anchored protein in cancer. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I. Citation Format: Hyunbum Jang, Mingzhen Zhang, Vadim Gaponenko, Ruth Nussinov. Comprehensive survey of KRas4B membrane interaction and orientation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2918.

Abstract CT033: KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors

Abstract Background: KRAS G12C oncogenic mutations occur in ~13% of non-small cell lung cancers (NSCLCs) and up to 4% of other solid tumors. JDQ443 is a selective, covalent, orally bioavailable, investigational KRASG12C inhibitor that irreversibly traps KRASG12C in the inactive, GDP-bound state. JDQ443 is structurally unique and forms novel interactions with KRAS in the switch II pocket. Methods: KontRASt-01 (NCT04699188) is a Phase Ib/II, open-label, multicenter, dose-escalation and dose-expansion trial of JDQ443 as monotherapy or in combination with TNO155 (SHP2 inhibitor) and/or tislelizumab (anti-PD-1 monoclonal antibody). Primary objectives of dose escalation are to assess safety and tolerability, and identify the maximum tolerated doses (MTDs) and/or recommended doses (RDs) and regimens for future studies. The primary objective of dose expansion is to assess efficacy. Key inclusion criteria: advanced, KRAS G12C-mutated solid tumors; previous standard-of-care treatment; age ≥18 yrs; ECOG PS 0-1. Key exclusion criteria for the JDQ443 monotherapy arm: active brain metastases, prior KRASG12C inhibitor treatment. Here, we present preliminary results for JDQ443 monotherapy dose escalation. Results: As of Nov 3, 2021, 39 pts were treated with JDQ443 PO continuously across 4 dose levels: 200 mg once daily (QD) (n=10), 400 mg QD (n=11), 200 mg twice daily (BID) (n=11), and 300 mg BID (n=7). Median age was 60 yrs (range 26-76), median prior lines of therapy was 3 (range 1-7), and indications included NSCLC (n=20) and colorectal cancer (CRC) (n=16). Median duration of exposure was 9.1 wks (range 0.9-21), with ongoing treatment in most pts (61.5%) at the time of cut-off. Treatment-related adverse events (TRAEs) occurred in 25 (64.1%) pts. Most TRAEs were Grade (Gr) 1-2. Four Gr 3 TRAEs occurred in 4 (10.3%) separate pts; there were no Gr 4-5 TRAEs. The most common TRAEs (occurring in ≥10% of pts) were fatigue (25.6%), nausea (15.4%), edema (12.8%), pruritus (10.3%), and vomiting (10.3%). There was one DLT (Gr 3 fatigue) and one treatment-related serious AE (Gr 3 photosensitivity reaction), each in separate pts treated at 300 mg BID. TRAEs led to dose reduction in 1 pt and discontinuation in 1 pt. A MTD was not reached. The RD was declared as 200 mg BID. At the RD, PK and PD modeling for JDQ443 predicted average KRASG12C target occupancy of >90% in >82% of pts. Using an efficacy cut-off date of Dec 13, 2021, for the 20 pts with NSCLC among the same 39 pts, the ORR (confirmed complete response or partial response) by RECIST 1.1 was 30.0% (6/20) across dose levels and 43.0% (3/7) at the RD. Additional data will be available at the time of presentation. Conclusions: JDQ443 demonstrates an acceptable safety and tolerability profile, with early signs of clinical activity in pts with NSCLC. Enrollment is ongoing to NSCLC and CRC dose-expansion groups for JDQ443 monotherapy at the RD, and to JDQ443 + TNO155 dose escalation. Citation Format: Daniel S. Tan, Toshio Shimizu, Benjamin Solomon, Rebecca S. Heist, Martin Schuler, Maria J. De Miguel Luken, Anas Gazzah, Martin Wermke, Christophe Dooms, Herbert H. Loong, Neeltje Steeghs, Enriqueta Felip, Conor E. Steuer, Eric van Cutsem, Ross A. Soo, Ashley C. Jaeger, Jaeyeon Kim, Kun Xu, Xueying Chen, Xiaoming Cui, Heather Burks, Anna Farago, Philippe A. Cassier. KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT033.

Abstract 4026: JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6

Abstract Background: Oncogenic mutations occurring in the KRAS component of the RAS/MAPK pathway slow nucleotide cycling between its active (GTP-bound) and inactive (GDP-bound) states, shifting it towards the active state and increasing oncogenic signaling. Among these mutations, the glycine-to-cysteine mutation of amino acid 12 (KRASG12C), found in ~13% of non-small cell lung cancer (NSCLC) and ~4% of colorectal cancer (CRC), can be specifically targeted and irreversibly locked in the inactive state by covalent modification of Cys12. We have previously reported the discovery and preclinical profile of JDQ443, a selective, oral, covalent inhibitor of KRASG12C that binds under the Switch II loop. Here, we report its antiproliferative and antitumor activity against panels of cancer cell lines as well as cell- (CDX) and patient-derived (PDX) tumor xenografts. Methods: JDQ443 antiproliferative activity was assessed by a high-throughput cell viability assay in a large panel of KRASG12C (n=17) and non-G12C (n=233) cell lines. Single-agent antitumor activity was assessed against a panel of KRASG12C CDX models from NSCLC (LU99, H2122, H2030, and HCC44), pancreatic (Mia PaCa-2), and esophageal (KYSE410) cancer cell lines, plus one non-G12C lung line (H441; KRASG12V). JDQ443 in vivo activity against a panel of KRASG12C NSCLC (n=10) and CRC (n=9) PDX models was assessed either as a single agent or in combination with TNO155 (SHP2 inhibitor [i]), trametinib (MEKi), or ribociclib (CDK4/6i). In vivo combination studies with TNO155 were also performed in CDX models (LU99, H2030, HCC44, and KYSE410). Results: In vitro, JDQ443 demonstrated potent antiproliferative activity selectively towards KRASG12C cell lines. Dose-dependent tumor growth inhibition/regression was observed for all KRASG12C CDX models, but not for the H441 KRASG12V model, and was independent of once-daily (QD) or twice-daily (BID) dosing. Single-agent antitumor activity (best average response, duration of reduction in tumor doubling time) was observed across both PDX panels and was improved by all three combination treatments. The JDQ443/TNO155 combination improved single-agent activity across CDX models, with comparable antitumor benefit maintained at QD or BID TNO155 schedules in two of three models (LU99 and KYSE410). Combination with TNO155 allowed a reduced dose of JDQ443 to achieve similar target occupancy and antitumor activity versus JDQ443 alone. Conclusions: JDQ443 demonstrates significant activity against a broad range of KRASG12C solid tumor models, both in vitro and in vivo, that is increased when combined with agents targeting both upstream and downstream components of the RAS signaling pathway. The combination benefit of JDQ443 + TNO155 over JDQ443 alone is maintained at reduced doses for both agents. Citation Format: Andreas Weiss, Hans Voshol, Diana Graus Porta, Carmine Fedele, Dario Sterker, Ruben De Kanter, Rowan Stringer, Toni Widmer, Alice Loo, Daniel A. Guthy, Kim S. Beyer, Nils Ostermann, Catherine LeBlanc, Marc Gerspacher, Andrea Vaupel, Richard Sedrani, Frederic Zecri, Saveur-Michel Maria, Francesco Hofmann, Peter Hammerman, Jeff Engelman, Edwige Lorthiois, Simona Cotesta, Saskia M. Brachmann. JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4026.

Abstract 2671: ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor models

Abstract SHP2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. SHP2 transduces upstream receptor tyrosine kinase (RTK) signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). The modulation of GEF activity impacts the rate at which KRAS cycles from the inactive GDP-bound state to the active GTP-bound state. ERAS-601 is a potent, selective small molecule allosteric inhibitor of SHP2. ERAS-601 potently inhibits the wild type SHP2 protein with a biochemical IC50 of 4.6 nM. ERAS-601 is a selective SHP2 inhibitor and demonstrates no appreciable inhibition against any off-target kinase or phosphatase across panels of 300 kinases and 12 phosphatases. ERAS-601 inhibits the loading of active GTP-bound oncogenic RAS and inhibits RAS/MAPK pathway signaling as measured by pERK1/2 inhibition and DUSP6 mRNA. ERAS-601 demonstrates anti-proliferative activity across a panel of human cancer cell line models with oncogenic alterations in the RAS/MAPK pathway. In a mouse in vivo study, ERAS-601 achieves substantial systemic exposure and demonstrates inhibition of ERK1/2 phosphorylation and DUSP6 mRNA levels in the NCI-H358 xenograft model. ERAS-601 also inhibits tumor growth in multiple RAS/MAPK-driven CDX and PDX models that harbor EGFR, KRAS, BRAF Class III, and NF1LOF mutations. ERAS-601 is a potent and selective allosteric SHP2 inhibitor that demonstrates anti-tumor activity in vitro and in vivo and is currently being studied as a monotherapy in an ongoing Phase 1 clinical study in patients with advanced or metastatic solid tumors (FLAGSHP-1, NCT04670679). Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Jennifer Yang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2671.

Abstract 3345: ERAS-601, a potent inhibitor of SHP2, synergistically enhances the activity of a FLT3 inhibitor, gilteritinib, in FLT3-mutated AML tumor models

Abstract ERAS-601 is a potent, small molecule allosteric inhibitor of wildtype SHP2, a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. ERAS-601 inhibits wildtype SHP2 biochemically with an IC50 of 4.6 nM and demonstrates selectivity across panels of 300 kinases and 12 phosphatases. SHP2 mediates upstream receptor tyrosine kinase (RTK) signaling via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). ERAS-601 inhibits the SHP2 dependent cycling of KRAS from the inactive GDP-bound state to the active GTP-bound state and demonstrates anti-proliferative activity in FLT3-mutated human cancer cells. Gilteritinib is a second-generation FLT3 inhibitor and has demonstrated clinical activity, but emerging resistance limits the benefit of monotherapy. Here, we explored the combination of gilteritinib with ERAS-601 in FLT3 mutated AML nonclinical models. The combination of ERAS-601 with gilteritinib inhibits the oncogenic RAS/MAPK signaling as measured by pERK1/2 and synergistically inhibits the cellular viability of FLT3-mutated AML cells in vitro. The combination of ERAS-601 with gilteritinib achieves a more durable tumor growth inhibition than the respective gilteritinib and ERAS-601 monotherapies in vivo. These preclinical data support the clinical development of ERAS-601 in combination with gilteritinib in FLT3-altered AML. Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent inhibitor of SHP2, synergistically enhances the activity of a FLT3 inhibitor, gilteritinib, in FLT3-mutated AML tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3345.

Abstract 2670: ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models

Abstract ERAS-601 is a potent, small molecule allosteric inhibitor of wildtype SHP2, a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. ERAS-601 inhibits wild type SHP2 biochemically with an IC50 of 4.6 nM and demonstrates selectivity across panels of 300 kinases and 12 phosphatases. SHP2 mediates upstream receptor tyrosine kinase (RTK) signaling via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). ERAS-601 inhibits the SHP2 dependent cycling of KRAS from the inactive GDP-bound state to the active GTP-bound state and demonstrates anti-proliferative activity in KRASG12C and EGFR amplified cell lines. The combination of upstream blockade of RAS-GTP cycling by ERAS-601 with inhibition of KRASG12C by a selective KRASG12C inhibitor synergistically inhibits cellular proliferation in multiple KRASG12C mutated human cancer cell lines. The combinations of ERAS-601 with KRASG12C inhibitors achieve tumor growth inhibition that is superior to the respective ERAS-601 and KRASG12C monotherapies in NSCLC and CRC CDX and PDX tumor models. Similarly, the combination of ERAS-601 with an EGFR antibody, cetuximab, inhibits oncogenic RAS/MAPK signaling as measured by pERK1/2 and enhances the anti-proliferative activity of cetuximab in RAS/RAF wild type CRC and HPV-negative HNSCC cell lines. The combination of ERAS-601 with cetuximab achieves tumor growth inhibition that is superior to respective ERAS-601 and cetuximab monotherapies in RAS/RAF wild type CRC and HPV-negative HNSCC CDX and PDX tumor models. These nonclinical data support the clinical development of ERAS-601 in combination with a KRASG12C inhibitor in NSCLC and CRC tumors, and ERAS-601 in combination with cetuximab in RAS/RAF wild type CRC as well as HPV-negative HNSCC tumors. Both combinations are being studied in ongoing clinical studies (HERKULES-2, NCT04959981; FLAGSHP-1, NCT04670679). Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2670.

The RAL Enigma: Distinct Roles of RALA and RALB in Cancer.

RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.

Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between “on” (GTP‐bound) and “off” (GDP‐bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP‐bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP‐bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD) of approximately 1 μM for the binding of these peptides to Gαs‐GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas‐mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs‐independent stimulation of adenylyl cyclase activity by forskolin. Protein‐protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi‐GTP in addition to Gαs‐GTP. In summary, we have identified a peptide inhibitor specific for Gαs‐GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically‐encoded probe or derivatized as a cell‐penetrating peptide to inhibit Gαs‐mediated cell signaling.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

Targeting KRAS Mutant in Non-Small Cell Lung Cancer: Novel Insights Into Therapeutic Strategies.

Although KRAS-activating mutations represent the most common oncogenic driver in non-small cell lung cancer (NSCLC), various attempts to inhibit KRAS failed in the past decade. KRAS mutations are associated with a poor prognosis and a poor response to standard therapeutic regimen. The recent development of new therapeutic agents (i.e., adagrasib, sotorasib) that target specifically KRAS G12C in its GDP-bound state has evidenced an unprecedented success in the treatment of this subgroup of patients. Despite providing pre-clinical and clinical efficacy, several mechanisms of acquired resistance to KRAS G12C inhibitors have been reported. In this setting, combined therapeutic strategies including inhibition of either SHP2, SOS1 or downstream effectors of KRAS G12C seem particularly interesting to overcome acquired resistance. In this review, we will discuss the novel therapeutic strategies targeting KRAS G12C and promising approaches of combined therapy to overcome acquired resistance to KRAS G12C inhibitors.

High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved ‘nested base-triple knot’ in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.

Turning Platelets Off and On: Role of RhoGAPs and RhoGEFs in Platelet Activity.

Platelet cytoskeletal reorganisation is a critical component of platelet activation and thrombus formation in haemostasis. The Rho GTPases RhoA, Rac1 and Cdc42 are the primary drivers in the dynamic reorganisation process, leading to the development of filopodia and lamellipodia which dramatically increase platelet surface area upon activation. Rho GTPases cycle between their active (GTP-bound) and inactive (GDP-bound) states through tightly regulated processes, central to which are the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs catalyse the dissociation of GDP by inducing changes in the nucleotide binding site, facilitating GTP binding and activating Rho GTPases. By contrast, while all GTPases possess intrinsic hydrolysing activity, this reaction is extremely slow. Therefore, GAPs catalyse the hydrolysis of GTP to GDP, reverting Rho GTPases to their inactive state. Our current knowledge of these proteins is constantly being updated but there is considerably less known about the functionality of Rho GTPase specific GAPs and GEFs in platelets. In the present review, we discuss GAP and GEF proteins for Rho GTPases identified in platelets, their regulation, biological function and present a case for their further study in platelets.

Delaying Emergence of Resistance to KRAS Inhibitors with Adaptive Therapy: “Treatment-to-Contain” Instead of “Treatment-to-Cure”

KRAS mutations are among the most common oncogenic abnormalities in cancer. Until recently, drug discovery pursuing KRAS did not produce therapeutic benefits for patients. Specific KRAS inhibitors, such as sotorasib and adagrasib, which bind covalently to codon 12 of substituted glycine to cysteine residue of the protein (G12C), have been approved by the FDA recently for the treatment of lung cancers. Binding of these drugs to the protein inhibits the activation of the GDP-bound inactive state to the GTP-bound active state. Phase 1/2 trials have shown potential anti-tumor activity, particularly in patients with previously treated non-small cell lung cancer. Acquired resistance, on the other hand, is inevitable, and the mechanisms include new KRAS mutations such as Y96D/C and other RAS-MAPK effector protein abnormalities. “Adaptive Therapy,” an ecologically inspired concept, focuses on extending the treatment-free period in a treatment course to delay the emergence of resistance. This review focuses on acquired mechanisms of resistance to KRAS G12C inhibitors, as well as the application of adaptive therapy in the treatment of KRAS-mutated patients to maintain acquired resistance sub-clones and extend progression-free survival.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography.

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.

Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division.

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

Resistance to KRASG12C Inhibitors in Non-Small Cell Lung Cancer.

KRAS mutations are one of the most prevalent oncogenic alterations in cancer. Until recently, drug development targeting KRAS did not convey clinical benefits to patients. Specific KRASG12C inhibitors, such as sotorasib and adagrasib, have been designed to bind to the protein’s mutant structure and block KRASG12C in its GDP-bound inactive state. Phase 1/2 trials have shown promising anti-tumor activity, especially in pretreated non-small cell lung cancer patients. As expected, both primary and secondary resistance to KRASG12C inhibitors invariably occurs, and molecular mechanisms have been characterized in pre-clinical models and patients. Several mechanisms such as tyrosine kinase receptors (RTKs) mediated feedback reactivation of ERK-dependent signaling can result in intrinsic resistance to KRAS target therapy. Acquired resistance to KRASG12C inhibitors include novel KRAS mutations such as Y96D/C and other RAS-MAPK effector protein mutations. This review focuses on the intrinsic and acquired mechanisms of resistance to KRASG12C inhibitors in KRASG12C mutant non-small cell lung cancer and the potential clinical strategies to overcome or prevent it.

Abstract P124: JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models

Abstract RAS is the most frequently mutated oncogene in cancer. KRAS G12C mutations are most prevalent in lung adenocarcinoma (~13%) and colorectal adenocarcinoma (~4%), and occur less commonly in other solid tumor malignancies. First generation KRASG12C inhibitors show anti-tumor activity in early phase clinical trials. However, the emergence of resistance, mediated at least in part by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways, limit the duration of response. Here we report the identification of JDQ443 (NVP-JDQ443), a novel KRASG12C inhibitor which binds under the switch II loop with a novel binding mode, exploiting unique interactions with the KRASG12C protein compared to sotorasib and adagrasib. JDQ443 potently inhibits KRASG12C cellular signaling and proliferation in a mutant selective manner by irreversibly trapping the GDP-bound state of KRASG12C through formation of a covalent bond with cysteine at position 12. Consistent with its mechanism as an irreversible inhibitor, JDQ443 shows sustained target occupancy (TO) in vivo (KRASG12C TO t1/2 ~ 66 h in the MiaPaCa2 model) despite a blood half-life of ~ 2 hours, and exhibits a linear PK/PD relationship. JDQ443 has dose-dependent anti-tumor activity in mice bearing KRAS G12C mutated tumor xenografts comparable to sotorasib and adagrasib. In mouse, rat, and dog, JDQ443 is orally bioavailable, achieves exposures in a range predicted to confer anti-tumor activity, and is well-tolerated. Continuous delivery of JDQ443 using mini-pump administration demonstrates that area under the curve (AUC), rather than maximal concentration (Cmax), is the driver of efficacy. Combination of JDQ443 with the SHP2 inhibitor TNO155 further increases KRAS G12C target occupancy in vivo, enhanced pre-clinical anti-tumor activity, and delayed the emergence of resistance in xenografts. A genome-wide CRISPR screen in 5 KRAS G12C mutated lung cancer cell lines identifies novel mechanisms of resistance to the KRAS/SHP2 drug combination. Furthermore, the characterization of JDQ443 alone and in combination with TNO155 in BaF/3 pools addicted to KRAS alleles that have previously been shown to mediate resistance to adagrasib in clinical samples will be discussed. Collectively, these data show that JDQ443 is a potent, mutant-selective, covalent irreversible KRASG12C inhibitor with favorable pharmaceutical properties. A phase Ib/II clinical trial of JDQ443 alone and in combination with TNO155 in patients with advanced solid tumors harboring the KRAS G12C mutation is ongoing (NCT04699188). Citation Format: Saskia M. Brachmann, Andreas Weiss, Daniel A. Guthy, Kim Beyer, Johannes Voshol, Michel Maira, Anirudh Prahallad, Diana Graus Porta, Christian Schnell, Nils Ostermann, Andrea Vaupel, Marc Gerspacher, Catherine Leblanc, Dirk Erdmann, Dario Sterker, Grainne Kerr, Giovannoni Jerome, Victoria Head, Rowan Stringer, Ruben De Kanter, Kearns Jeff, Danielle Roman, Toni Widmer, Peter Wessels, Eloisa Jimenez Nunez, Richard Sedrani, Frederic Zecri, Francesco Hofmann, Jeff Engleman, Edwige Lorthiois, Simona Cotesta. JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P124.

Towards resolution of a paradox in plant G-protein signaling.

G-proteins are molecular on-off switches that are involved in transmitting a variety of extracellular signals to their intracellular targets. In animal and yeast systems, the switch property is encoded through nucleotides: a GDP-bound state is the "off-state" and the GTP-bound state is the "on-state". The G-protein cycle consists of the switch turning on through nucleotide exchange facilitated by a G-protein coupled receptor and the switch turning off through hydrolysis of GTP back to GDP, facilitated by a protein designated REGULATOR OF G SIGNALING 1 (RGS). In plants, G-protein signaling dramatically differs from that in animals and yeast. Despite stringent conservation of the nucleotide binding and catalytic structures over the 1.6 billion years that separate the evolution of plants and animals, genetic and biochemical data indicate that nucleotide exchange is less critical for this switch to operate in plants. Also, the loss of the single RGS protein in Arabidopsis (Arabidopsis thaliana) confers unexpectedly weaker phenotypes consistent with a diminished role for the G cycle, at least under static conditions. However, under dynamic conditions, genetic ablation of RGS in Arabidopsis results in a strong phenotype. We explore explanations to this conundrum by formulating a mathematical model that takes into account the accruing evidence for the indispensable role of phosphorylation in G-protein signaling in plants and that the G-protein cycle is needed to process dynamic signal inputs. We speculate that the plant G-protein cycle and its attendant components evolved to process dynamic signals through signaling modulation rather than through on-off, switch-like regulation of signaling. This so-called change detection may impart greater fitness for plants due to their sessility in a dynamic light, temperature, and pest environment.