Mitochondrial function relies on close coordination between the mitochondrial and nuclear genomes. Disruption to this coordination-via mitonuclear mismatch-can impair metabolic efficiency, particularly under energetically demanding conditions such as during development. The nutritional environment further modulates mitochondrial demands, suggesting that mitonuclear genotype and diet may interact to shape life-history traits and behaviour. Here, we investigate how early-life diet and mitonuclear genotype jointly influence development time, adult body size, and nutritional preference in Drosophila melanogaster. Using a full-factorial panel of putatively matched and mismatched combinations (cybrids) of mitonuclear genotype derived from natural Australian populations, we reared flies on diets varying in their ratio of macronutrients and assessed how this influenced larval development and subsequent adult diet preference. Developmental rate was significantly influenced by mitonuclear coevolution and diet, with cybrids showing delayed development under all conditions, with dietary extremes exacerbating this effect. Despite this, egg-to-adult viability remained unaffected. Adult nutritional behaviour exhibited clear genotype- and diet-dependent effects. Flies reared on high-protein diets increased carbohydrate intake as adults, while those reared on high-carbohydrate diets increased protein intake, suggesting compensatory feeding responses. Mitonuclear mismatch further modulated nutrient consumption, particularly in females, whose carbohydrate intake was influenced by intergenomic compatibility and early-life dietary conditions. Males' protein consumption was also impacted by mitonuclear coevolution across all developmental diets. Finally, body size was also shaped by interactions between mitonuclear genotype and diet. Together, our findings demonstrate that mitonuclear compatibility and the composition of the early nutritional environment interact to shape developmental and behavioural phenotypes. These results support a role for mitonuclear coadaptation in mediating metabolic plasticity, highlighting the evolutionary and physiological significance of genotype-specific mitonuclear coordination.

Abstract Introduction: Addiction to the MAPK pathway drives a large proportion of cancers, and pancreatic tumors almost universally display RAS mutations. IMM-1-104, a once-daily oral treatment being evaluated in a Phase 1/2a trial for RAS-mutant solid tumors [NCT05585320], offers a novel deep cyclic inhibition (DCI) approach in targeting the MAPK pathway at MEK. Traditionally, MAPK-targeted drugs inhibit the pathway chronically causing serious class-effect toxicities and limited durability due to resistance. In contrast, IMM-1-104’s unique pharmacokinetic (PK) profile was designed to drive pulsatile MEK inhibition, with the goal of improving tolerability and providing more durable activity across a broad range of MAPK-driven tumors. Phase 1 dose escalation of IMM-1-104 revealed no dose-limiting toxicities, high oral bioavailability, plasma half-life of approximately 2-hours, and pharmacodynamic (PD) data supporting DCI of the MAPK pathway. Experimental Procedures: IMM-1-104 responses in humanized 3D tumor growth assays (3D-TGA) were combined with NGS data using machine learning (ML) to refine a pharmacogenomic response model. Evaluation of databases such as AACR Project GENIE enabled prediction of patient alignment of preclinical models based on genomic profile, identification of patient populations displaying MAPK pathway addiction and projected sensitivity to IMM-1-104 mono- or combination therapy. To test combinations with approved chemotherapy agents, IMM-1-104, gemcitabine (GEM) and nab-paclitaxel (PAC) and 5-fluorouracil (5FU) were evaluated in tumor xenograft models with drugs alone or across multiple combinations. Summary of New Data: IMM-1-104 showed promising combination effects when treated with GEM or PAC in 3D-TGA pancreatic cancer models. In a MIA PaCa-2 tumor xenograft model, IMM-1-104 alone showed greater tumor growth inhibition (TGI) than any single or combination chemotherapy tested. Further, combinations of IMM-1-104 plus chemotherapy resulted in near complete responses in a majority of animals. At day 39, antitumor activity (TGI%) was 103% for IMM-1-104 at 125 mg/kg BID PO, 25.2% for GEM at 60 mg/kg IP Q4D, 62.2% for PAC at 10 mg/kg IV Q4D and 36.6% for 5FU at 50 mg/kg IP Q4D. Based on these results and additional 3D-TGA pharmacogenomics data, ML modeling was advanced to query the GENIE database and identify biomarkers of response and resistance with the goal of further informing mono and combination treatment options with IMM-1-104 in pancreatic cancer. Conclusions: The Phase 2a portion of the ongoing IMM-1-104 clinical study includes five arms, three of which focus on patients with pancreatic cancer, where IMM-1-104 will be evaluated as both monotherapy and in select combinations with approved chemotherapeutic agents. The new in vitro, in vivo and ML modeling data presented here further support an advancing translational roadmap for IMM-1-104 in pancreatic cancer. Citation Format: Peter King, Jason Funt, Sarah Kolitz, Praveen Nair, Jan de Jong, Amy Yamamura, Mai Johnson, Jenny Zhang, Kevin Fowler, Anna Travesa, Amy Axel, Chris Walker, Benjamin J. Zeskind, Brett M. Hall. Activity of IMM-1-104 alone or in combination with chemotherapy in RAS-altered pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4195.

Abstract Introduction: IMM-1-104, with pan-RAS activity through deep cyclic inhibition MEK, was evaluated in humanized 3D preclinical tumor models displaying diverse MAPK pathway activation events. Based on drug-response, sensitivity and resistance profiles, a biomarker signature for IMM-1-104 was developed in order to project potential therapeutic response of cancer patients found in the AACR Project GENIE (GENIE) database. Experimental Procedures: Humanized 3D preclinical models better predict in vivo tumor responses versus 2D culture and more accurately replicate biology of human tumors. Therefore, the antitumor activity of IMM-1-104 was evaluated in over 130 tumor models spanning 12 distinct histologies in the humanized 3D tumor growth assay (3D-TGA). Cell-based whole exome sequencing readouts were combined with 3D-TGA results to build a pharmacogenomic response algorithm. When applied to the GENIE patient database, resultant tumor-specific response landscapes helped to inform an early pan-RAS clinical trial design for IMM-1-104. Summary of New Data: A machine learning model was developed to predict IMM-1-104 sensitivity using response-associated genes and signaling networks that were identified using 3D-TGA pharmacogenomics data. This model was used to estimate GENIE patient IMM-1-104 response profiles across key solid tumor indications. In addition, mutation constellations from GENIE were compared with those observed in cell lines to identify preclinical models that best resemble real-world patients. This effort was designed to further enrich the translational fidelity of specific tumor models with the goal of translationally identifying patient populations most likely to benefit from IMM-1-104 treatment. Conclusions: The depth of response to IMM-1-104 was evaluated across a panel of diverse 3D-TGA tumor models and led to identification of a biomarker signature for therapeutically addressable MAPK pathway addiction. To translate these findings into a relevant clinical application, a response algorithm was developed and applied to the GENIE database, which has cataloged the molecular profiles of over 100,000 cancer patients. Mutational landscapes of patients within GENIE helped identify preclinical models that better represent patient profiles likely to be encountered in the clinic. This approach could, as a general principle, be applied as a tool for improving biomarker discovery and clinical translation of oncology drugs. Citation Format: Praveen Nair, Sarah Kolitz, Jason Funt, Peter J. King, Kevin D. Fowler, Anna Travesa, Ian Rose, John Brothers, Amy Axel, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Humanized 3D tumor models that are mutationally aligned with AACR GENIE patients predict IMM-1-104 activity in RAS-addicted tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4265.

Abstract Introduction: Novel dual-MEK inhibitor IMM-1-104 is under clinical investigation for use in RAS-addicted solid tumors. Approved KRAS G12C inhibitors are available but cover a limited subset of patients. For example, the KRAS G12C substitution occurs in only 1-3% of pancreatic cancers. We assessed response to IMM-1-104 across RAS mutant preclinical models to determine whether a preference was observed for mutation position, or for specific amino acid substitutions. Experimental Procedures: Response to IMM-1-104 was measured in a humanized 3D tumor growth assay across 133 tumor models. Sixty-nine of these models have a reported RAS mutation, and all models are being mutationally profiled by whole exome sequencing, with the majority (~75%) completed to date. The RAS-mutant panel spans 11 tissue types and includes a subset of 30 confirmed KRAS G12 mutated cell lines drawn from three major indications: 12 pancreatic, 11 lung, and 7 colorectal cancer models. Based on the 3D assay, cell lines were classified into sensitive, intermediate, and resistant to IMM-1-104. The distribution of responses was then assessed across mutation position and amino acid substitutions. Summary of New Data: In the full dataset across 69 RAS-mutant models, at least one model displayed response to IMM-1-104 (sensitive or intermediate) for each observed mutation in K/N/HRAS. That is, no particular mutation position or amino acid substitution was exclusively found to be resistant. Association of response with amino acid identity was further evaluated in a subset of lines for the most frequently altered residue in KRAS, G12. A distribution of responses was observed for each amino acid substitution. The G12 substitutions having at least four cell lines each total across the three indications included G12C (8 lines), D (5 lines), R (4 lines), and V (11 lines). Across cell lines for each of these substitutions, multiple response categories were observed. In each case, half or more lines fell into the intermediate category with the rest falling into one or both of the other response categories. For example, out of the 8 KRAS G12C lines, 6 showed intermediate response, 1 showed resistance, and 1 showed sensitivity. Examining these distributions together, no significant statistical relationship was seen between the amino acid substitution and response categories by Fisher’s exact test. Conclusions: Across 69 RAS-mutated cell lines, each mutation position or amino acid substitution was associated with sensitive or intermediate response in at least one line. Looking at the most commonly mutated position in KRAS, across 30 KRAS G12 mutated cell lines from three cancer indications, no significant preference was observed with respect to response to IMM-1-104 for a particular amino acid at G12. These observations suggest potential relevance of IMM-1-104 to a broad RAS-driven patient population. Citation Format: Sarah Kolitz, Praveen Nair, Mai Johnson, Jason Funt, Peter J. King, Kevin D. Fowler, Anna Travesa, Frank Wang, John Brothers II, Amy Axel, Scott Barrett, Benjamin J. Zeskind, Brett Hall. Pan-RAS IMM-1-104 activity in humanized 3D tumor models is independent of specific amino acid substitution [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B021.

Abstract Introduction: Many tumors are addicted to MAPK pathway activation, including the >20% of human tumors with mutations in RAS or RAF1. IMM-1-104 is an oral once-daily treatment currently in Phase 1 in patients with RAS-mutant solid tumors [NCT05585320]. To date, drugs disrupting the MAPK pathway have done so chronically, leading to dose-limiting toxicities (DLTs) and poor response durability. In contrast, IMM-1-104 was designed to provide deep cyclic inhibition (DCI) of the MAPK pathway via a unique pharmacokinetic (PK) profile with high peak plasma drug levels and a near zero drug trough between doses. This promotes pulsatile inhibition of MEK, depriving tumors of sustained signaling of a critical oncogenic pathway while limiting toxicity and durability issues associated with chronic MEK inhibition. In Phase 1a dose escalation, no DLTs were observed, the plasma drug half-life was ~2-hours, and pharmacodynamic (PD) data were consistent with DCI. Phase 1b dose expansion is underway. Translational efforts are focused on identifying MAPK pathway addiction and sensitivity to IMM-1-104. Tumor models displaying patient-aligned genomic profiles against large patient databases such as AACR Project GENIE1 were tested in humanized 3D tumor growth assays (3D-TGA). Computational modeling based on response and in-house genomic data was used to inform identification of patient populations for IMM-1-104 monotherapy and potential combination opportunities. Experimental Procedures: Using cancer-specific, patient-aligned cell lines, IMM-1-104 activity was characterized in the 3D-TGA. Whole exome sequencing was performed to confirm alteration status, and a further subset subjected to RNA sequencing. Pharmacogenomic data were used to generate a model predictive of response to IMM-1-104 and identify biomarker-aligned patient subpopulations. Selected model predictions were then tested in subcutaneous tumor xenograft models in female BALB/c nude mice. Summary of New Data: Assessment of IMM-1-104 across >190 patient-aligned models demonstrated diverse responses across a wide range of MAPK-driven tumor types, including those with RAS or RAF mutations. In addition to RAS, these data suggested additional potential for IMM-1-104 in BRAF-mutant disease. Therefore, IMM-1-104 was tested alone and with encorafenib in the HT-29 colorectal BRAFV600E mutant xenograft model. Monotherapy with either encorafenib or IMM-1-104 displayed superior tumor growth inhibition to binimetinib. IMM-1-104 in combination with encorafenib drove deeper regressions and superior durability of response in a head-to-head in vivo comparison versus binimetinib plus encorafenib. Conclusions: We used an integrated platform of translational experiments and informatics to identify patient-aligned model systems, prioritize factors relevant for response to IMM-1-104’s unique DCI profile, and elucidate combination opportunities to potentially inform clinical development strategies. Citation Format: Praveen Nair, Sarah Kolitz, Jason Funt, Jan de Jong, Peter King, Amy Yamamura, Mai Johnson, Jenny Zhang, Kevin D Fowler, Anna Travesa, Amy Axel, Chris Walker, Scott Barrett, Benjamin J Zeskind, Brett Hall. Predicting activity of IMM-1-104 as single agent and in combination for patients with RAS or RAF mutant tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A134.

Introduction: A significant proportion of cancer patients have tumors addicted to uncontrolled MAPK signaling. Activating mutations in RAS or RAF are often directly responsible and have been observed in over 20% of human tumors1. Because MEK is downstream of RAS and RAF, it is an appealing drug target. However, MEK inhibitors have historically suffered from poor clinical durability, high toxicity and a susceptibility to pathway reactivation that has limited monotherapy activity in the RAS mutant setting. Unlike other MEK inhibitors, IMM-1-104 [NCT05585320] and IMM-6-415 are designed with distinctive features including both (1.) a unique target engagement mechanism that helps resist MAPK pathway reactivation and (2.) a pharmacokinetic (PK) profile that enables fast cadence deep cyclic inhibition (DCI). DCI drives pulsatile targeted inhibition that deprives tumor cells of a critical oncogenic pathway while limiting drug-related toxicities by affording normal cells an adequate PK recovery window between drug doses. To our knowledge, IMM-6-415’s preclinical activity is driven by the shortest drug plasma half-life (0.3-hours in mice) of any MEK inhibitor developed to date. Experimental Procedures: IMM-6-415 has already demonstrated promising activity in RAS-mutant xenograft tumor models (SITC 2022). Here, the antitumor activity of IMM-6-415 was evaluated in over 60 humanized 3D tumor growth assays (3D-TGA), which included 30 BRAF class I-mutant tumor models. Additionally, multiple drug-drug combinations have been explored, including vertical drug combinations with BRAF inhibitors. IMM-6-415, binimetinib and encorafenib were tested head-to-head as single agents and in combination with encorafenib in BRAFV600E melanoma and colorectal subcutaneous tumor xenograft models in female BALB/c nude mice. Summary of New Data: As monotherapy, IMM-6-415 demonstrated antitumor activity in over 50% (34 of 66) of the 3D-TGA models tested, including 30 BRAF-mutant preclinical models in which 19 (63%) showed activity. Therefore, we further explored both monotherapy and combination activity of IMM-6-415 in the A-375 (melanoma) and HT-29 (colorectal) BRAF V600E tumor xenograft models. Monotherapy treatment with encorafenib or IMM-6-415 displayed superior tumor growth inhibition (TGI) when compared to binimetinib. Furthermore, the combination of IMM-6-415 plus encorafenib prompted greater TGI with superior durability of response when tested head-to-head against the combination of binimetinib plus encorafenib in vivo at human equivalent doses for registered drugs. Conclusions: IMM-6-415 demonstrated promising activity and tolerability in preclinical models alone and in combination with encorafenib. In combination with encorafenib, IMM-6-415 achieved a greater TGI in vivo than the combination of encorafenib plus binimetinib in BRAFV600E colorectal cancer and melanoma tumor models, suggesting an opportunity for IMM-6-415 as monotherapy or in combination in BRAF mutant tumors. Citation Format: Anna Travesa, Mai Johnson, Peter King, Praveen Nair, Jason Funt, Sarah Kolitz, Kevin D Fowler, John Brothers II, Amy Axel, Scott Barrett, Benjamin J Zeskind, Brett Hall. Deep Cyclic Inhibition of the MAPK pathway with IMM-6-415, alone and in combination with encorafenib, demonstrates anti-tumor activity and tolerability in RAF mutant tumors in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A093.

e15084 Background: Elevated RAS-RAF-MEK-ERK (MAPK pathway) signaling is observed in over half of all solid human tumors, and mutations in RAS or RAF account for a large fraction. Given MEK’s unique position in the MAPK cascade, it remains an attractive target in cancer. However, FDA-registered MEK inhibitors are susceptible to pathway reactivation events that limit their use to RAF mutant disease and cause on-target toxicities stemming from chronic target engagement. IMM-1-104 is a novel, allosteric dual-MEK inhibitor designed for better applicability to RAS mutant tumors by preventing MEK reactivation. Endowed with a short plasma half-life, IMM-1-104 promotes deep cyclic inhibition with a near-zero drug trough, affording normal cells a chance to recover between doses. Methods: We characterized IMM-1-104’s pharmacologic activity across 52 tumor cell lines that spanned 11 distinct tumor types in a humanized, ECM-based 3D tumor growth assay (3D-TGA). The 3D-TGA has better predicted in vivo tumor responses versus 2D culture and more accurately reflects human tumor biology. Tumor models were categorized based on in vivo drug PK limits as sensitive to IMM-1-104 (EC50 < 1uM), intermediate (1uM≤EC50≤10uM and ≥25% inhibition at 10uM) or resistant otherwise. Models were evaluated by whole exome sequencing, along with RNA sequencing in the 3D context, to profile determinants of sensitivity and resistance and to prioritize patient populations most likely to respond to IMM-1-104. Results: Models sensitive to IMM-1-104 were enriched for MAPK driver mutations, consistent with pathway addiction. We reasoned that activation of parallel compensatory pathways that can reduce reliance on MAPK signaling may increase the likelihood of resistance to IMM-1-104. Pathways and genes suspected of contributing to resistance helped refine signatures based on 3D-TGA outcome data. Models with a MAPK driver mutation and compensatory mutations such as PIK3CA or PTEN deletion were more likely to show intermediate response than those with a greater addiction to MAPK drivers. Models lacking a clear MAPK driver mutation but harboring other putative resistance alterations were more likely to be resistant in the 3D-TGA. Conclusions: To better understand the relevance of tumor model responses in the 3D-TGA relative to RAS mutant patient populations, we computationally compared tumor model data to patient somatic alterations, identified in the public resource GENIE, which has cataloged the molecular profiles of over 100,000 cancer patients. Based on model-to-patient molecular mapping, we identified biomarker-defined subsets of sensitive KRAS mutant lung and colorectal models. The most broadly sensitive patient-aligned models in the 3D assay were KRAS mutant pancreatic cancer and NRAS mutant melanoma patients, supporting the inclusion of such patients in planned clinical studies of IMM-1-104.

Background KRAS is the most frequently altered RAS gene (~85%) and is often mutated in pancreatic ductal adenocarcinoma (PDAC; 95%), non-small cell lung cancer (NSCLC; 40%) and colorectal cancer (CRC; 45%). KRAS-G12C inhibitors (sotorasib/adagrasib) have demonstrated single-agent activity in all three tumor types. However, acquired resistance and limited biomarker positive patients (e.g., only 1-3% of PDAC and CRC) limit broader access and overall response to G12C inhibitors, prompting evaluation of combination partners including immune therapies. In contrast to G12C-mutant focused KRAS inhibitors, MEK inhibitors may broaden the potential for immune therapy in RAS-mutant tumors but have largely proven ineffective in this setting. Methods IMM-6-415 is a novel, third-generation dual MEK inhibitor that reduces both pMEK and pERK in RAS-mutant tumor models at sub-100 nM potencies. IMM-6-415 drug-like properties have been evaluated across a series of preclinical in vitro and in vivo models focusing on activity in those with mutant RAS. Cell-based 2D and 3D biochemical and pharmacologic assays were performed across multiple models, and several in vivo studies have been completed, including: (1.) A549 (KRAS-G12S NSCLC) xenograft model, (2.) Colon-26 (KRAS-G12D CRC) syngeneic model, (3.) CT-26 (KRAS-G12D) syngeneic model. The CT-26 in vivo study evaluated both single-agent IMM-6-415 and combinations with PD1 or CTLA4 checkpoint inhibitors. Results IMM-6-415 reduced pERK and pMEK across all RAS mutant models tested. Humanized 3D tumor models revealed a promising sensitivity profile for IMM-6-415 in RAS-mutant CRC and PDAC. The maximum tolerated dose (MTD) of IMM-6-415 was 175 to 180 mg/kg BID PO from the Colon-26 (96.4% TGI) and A549 (93.9% TGI) studies, yet the optimal MEKio combination dose/schedule was 120 mg/kg BID PO in the CT-26 study. At 28 days treatment, 33% (4/12) mice remained on study in either the (10 mg/kg BIW IP) anti-PD1 or anti-CTLA4 alone treated groups, whereas 58% (7/12) mice remained in the IMM-6-415 treatment arm at 120 mg/kg BID PO. However, 92% (11/12) and 83% (10/12) mice remained in the IMM-6-415 plus anti-PD1 or anti-CTLA4 combination at the same doses, respectively. Conclusions We demonstrate that IMM-6-415 displays single-agent activity in multiple RAS-mutant models, has a 0.3h half-life, is well tolerated in mice, and when combined at sub-MTD levels with either PD1 or CTLA4 checkpoint inhibitors, significantly improved responses in the CT-26 model (p-value < 0.05). Our data suggest that moderated, cyclic inhibition of the MAPK pathway in RAS mutant tumors is active and may enhance therapeutic activity of immune checkpoint inhibitors. Ethics Approval The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to execution. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

e16297 Background: KRAS mutations are common in pancreatic ductal adenocarcinoma (PDAC). While 90% of PDAC tumors display activating mutations in KRAS, only ̃2% are G12C, a specific KRAS mutation targeted by inhibitors such as sotorasib or adagrasib. MEK, which lies downstream of KRAS, is an attractive target to more broadly counteract elevated MAPK signaling regardless of the upstream mutation. However, FDA registered MEK inhibitors are prone to pathway reactivation events, which limit their utility in RAS mutant disease and necessitate chronic pathway inhibition that contributes to on-target toxicity. In contrast, IMM-1-104 is a novel, allosteric dual-MEK inhibitor designed to block pathway reactivation by disrupting phosphorylation of both MEK and ERK and has a short plasma drug half-life. These characteristics enable IMM-1-104 to drive deep cyclic MAPK pathway inhibition, with the potential to inhibit tumors driven by diverse RAS mutations. Methods: IMM-1-104 was tested head-to-head versus sotorasib, adagrasib, selumetinib and binimetinib in a series of preclinical models to characterize differential activity of each compound against tumors driven by diverse KRAS mutations. Cell-based 2D biochemical and 3D growth assays were performed across nine PDAC models, and the Capan-2 PDAC xenograft animal model was used to evaluate single agent activity of IMM-1-104 (75, 100, 150 mg/kg BID p.o. or 150 mg/kg QD p.o.) vs. sotorasib or adagrasib (30 and 100 mg/kg QD p.o. each) for 21 days treatment after tumors had reached volumes of 150 to 200 mm3. Results: IMM-1-104 alone led to reductions in both pERK and pMEK across all 9 PDAC models tested (KRAS status shown), including Hs766T (Q61H), MIA PaCa-2 (G12C), Capan-2 (G12V), AsPC-1 (G12D), CFPAC-1 (G12V), BxPC3 (wild type), Panc 10.05 (G12D), Capan-1 (G12V) and PSN1 (G12R). A head-to-head comparison in vivo demonstrated no Tumor Growth Inhibition (TGI) by sotorasib and adagrasib in KRAS-G12V mutant Capan-2 PDAC tumors, while IMM-1-104 prompted TGIs of 49 to 84% across all doses and schedules tested. Conclusions: Despite multiple clinical studies, including Phase 2 studies for the MEK inhibitors trametinib and selumetinib, limited progress has been made in PDAC treatment since FOLFIRINOX’s approval in 2011. The Phase 2 KRYSTAL-1 and Phase 1/2 CodeBreaK 100 studies recently reported promising activity in KRAS-G12C PDAC, suggesting an opportunity for disruption of KRAS addiction. IMM-1-104 and sotorasib previously demonstrated comparable tumor regressions in vivo in a KRAS G12C mutant model, MIA PaCa-2 (2021 EORTC). Examining the broader activity of IMM-1-104 across 9 PDAC tumor models yielded data suggesting that deep, cyclic MEK inhibition by IMM-1-104 has the potential to offer a unique advantage over first generation MEK inhibitors and KRAS-G12C inhibitors in PDAC by inhibiting tumors driven by a broader range of more common KRAS mutations.

Abstract Background: Inappropriate activation of the MAPK pathway, often stemming from mutations in RAS or RAF, represents one of the most common oncogenic events in human cancer. Therefore, MEK1 and MEK2 (MEK), which lie downstream of RAS and RAF but upstream of ERK, have represented an attractive drug target for over two decades. Unfortunately, first generation MEK inhibitors have been limited by pathway reactivation events and serious on-target drug toxicities that restrain clinical utility, especially in patients with RAS mutant tumors. We sought to develop a new approach to MEK inhibition that would be more effective in RAS mutant tumors and with improved tolerability. IMM-1-104 is a novel dual-MEK inhibitor that is designed to disrupt phosphorylation of MEK and subsequently prevent activation of ERK1 and ERK2 (ERK). IMM-1-104’s dual-MEK mechanism resists RAF activation of MEK, and its short drug half-life allows for chronic dosing while maintaining a near-zero drug trough for improved tolerability. Materials & Methods: IMM-1-104 was profiled across a series of preclinical experiments to assess its physicochemical and drug-like properties. Cell-free and cell-based in vitro as well as in vivo characteristics were evaluated, including four independent in vivo pharmacology rodent studies in lung, colon and skin tumor models. Results: IMM-1-104 is a highly selective, orally bioavailable, non-ATP competitive, allosteric dual-MEK inhibitor. At drug exposures up to 1 micromolar (uM) in cell-free and in situ kinome screens, thermodynamic interactions and altered activity levels were observed for MEK1 and MEK2. At higher drug exposures up to 10 uM, IMM-1-104 also thermodynamically interacted with RAF1 (CRAF) and prompted reduction of KSR1 and KSR2 activation markers. IMM-1-104 displayed dual-MEK activity (i.e., RAS mutant tumor cell reductions in both ERK and MEK phosphorylation) across multiple human tumor cell-based models including A549 (KRAS-G12S) lung, A375 (BRAF-V600E) and SK-MEL-2 (NRAS-Q61R) melanoma. Pharmacokinetic studies in rodent models revealed a drug plasma half-life for IMM-1-104 of approximately 1.3 hours. In vivo tumor pharmacology studies in A549 and A375 achieved tumor stasis (i.e., 85% to 95% Tumor Growth Inhibition) with regressions observed in Colon-26 and SK-MEL-2. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has proven to be broadly active and well-tolerated in multiple RAS and RAF mutant preclinical tumor models in rodents. IMM-1-104 drives deep, cyclic inhibition of the MAPK pathway (improving tolerability) while resisting pathway bypass mechanisms (improving activity). Our collective data suggest that RAS and RAF mutant tumor cells are not able to tolerate periods of deep but cyclic MAPK pathway inhibition. Overall, these results are consistent with on and off signaling events that can significantly impact cell fate decisions (i.e. signaling dynamics). Citation Format: Peter J. King, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. IMM-1-104: a novel, oral, selective dual-MEK inhibitor that displays broad antitumor activity and high tolerability across RAS and RAF mutant tumors in vivo [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 P252.

The amount of genetic variation for fitness within populations tends to exceed that expected under mutation-selection-drift balance. Several mechanisms have been proposed to actively maintain polymorphism and account for this discrepancy, including antagonistic pleiotropy (AP), where allelic variants have opposing effects on different components of fitness. Here, we identify a non-coding indel polymorphism in the fruitless gene of Drosophila melanogaster and measure survival and reproductive components of fitness in males and females of replicate lines carrying each respective allele. Expressing the fruitless region in a hemizygous state reveals a pattern of AP, with one allele generating greater reproductive fitness and the other conferring greater survival to adulthood. Different fitness effects were observed in an alternative genetic background, which may reflect dominance reversal and/or epistasis. Our findings link sequence-level variation at a single locus with complex effects on a range of fitness components, thus helping to explain the maintenance of genetic variation for fitness. Transcription factors, such as fruitless, may be prime candidates for targets of balancing selection since they interact with multiple target loci and their associated phenotypic effects.

The maternal inheritance of mitochondrial genomes entails a sex-specific selective sieve, whereby mutations in mitochondrial DNA can only respond to selection acting on females. In theory, this enables male-harming mutations to accumulate in mitochondrial genomes as long as they are neutral, beneficial, or only slightly deleterious to females. Ultimately, this bias could drive the evolution of male-specific mitochondrial mutation loads, an idea known as mother's curse. Earlier work on this hypothesis has mainly used small Drosophila panels, in which naturally sourced mitochondrial genomes were coupled to an isogenic nuclear background. The lack of nuclear genetic variation in these designs has precluded robust generalization. Here, we test the predictions of mother's curse using a large Drosophila mitonuclear genetic panel, comprising nine isogenic nuclear genomes coupled to nine mitochondrial haplotypes, giving a total of 81 different mitonuclear genotypes. Following a predictive framework, we tested the mother's curse hypothesis by screening our panel for wing size. This trait is tightly correlated with overall body size and is sexually dimorphic in Drosophila. Moreover, growth is heavily reliant on metabolism and mitochondrial function, making wing size an ideal trait for the study of the impact of mitochondrial variation. We detect high levels of mitonuclear epistasis, and more importantly, we report that mitochondrial genetic variance is larger in male than female Drosophila for eight out of the nine nuclear genetic backgrounds used. These results demonstrate that the maternal inheritance of mitochondrial DNA does indeed modulate male life history traits in a more generalisable way than previously demonstrated.

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (<1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials & Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [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 P240.

Abstract We developed a novel dual MEK inhibitor, IMM-1-104, that showed 94% tumor growth inhibition (TGI) in the syngeneic C26 mouse model through 10 days at 150 mg/kg (mpk) BID, with only 3.8% tumor-adjusted body weight loss (BWL) through 24 days. The mechanism differs from that of standard MEK inhibitors (MEKi), and leverages signaling dynamics to decouple efficacy and toxicity. IMM-1-104 achieves deep cyclic inhibition of the pathway rather than constant blockade, and prevents the RAF1 (CRAF) bypass which has hampered standard MEKi. In this study, we examined the transcriptional effects of IMM-1-104 in the C26 model via RNA sequencing. These observations confirmed the pattern of deep cyclic inhibition, demonstrating strong MAPK pathway inhibition in tumor 2h after treatment, and near complete release 12h following treatment. This pattern was observed both after the initial dose as well as following chronic BID dosing (18 days), indicating that deep cyclic inhibition was sustainable across chronic dosing. Filtering the transcriptional signatures to examine effects that followed the pattern of deep cyclic inhibition (strong effect at 2h and release at 12h) revealed effects on mitochondrial and metabolism genes. These genes included Hk2, which was downregulated more than 2-fold after 2h of treatment with 100 mpk IMM-1-104 both after the initial dose and following chronic BID dosing. The effect on Hk2 occurred in tumor but not in muscle tissue from the same mice. These results were independent of adaptive immunity, as they were also observed in nude mice. Top transcriptional effects of IMM-1-104 treatment in tumor also included an increase in expression of Wnt pathway genes at 2h, following either a single dose or chronic BID dosing, at both 25 and 100 mpk. This increase was also observed at 12h following the 100 mpk dose. Activation of Wnt signaling in CRC tumors has previously been reported with standard MEK inhibitors. The transcriptional profile of IMM-1-104 in the C26 model highlighted key aspects of its mechanism related to signaling dynamics, which helps explain its ability to achieve high TGI and tolerability. The profile also highlighted aspects shared with standard MEKi that could shed light on potential resistance pathways. Citation Format: Sarah E. Kolitz, Kevin D. Fowler, Peter J. King, Benjamin J. Zeskind, Brett M. Hall. Transcriptional effects in C26 tumor highlight mechanistic aspects of a novel dual MEK inhibitor, IMM-1-104 [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 P254.

Meiotic drive systems are associated with low-frequency chromosomal inversions. These are expected to accumulate deleterious mutations due to reduced recombination and low effective population size. We test this prediction using the 'sex-ratio' (SR) meiotic drive system of the Malaysian stalk-eyed fly Teleopsis dalmanni. SR is associated with a large inversion (or inversions) on the X chromosome. In particular, we study eyespan in males carrying the SR chromosome, as this trait is a highly exaggerated, sexually dimorphic trait, known to have heightened condition-dependent expression. Larvae were raised in low and high larval food stress environments. SR males showed reduced eyespan under the low and high stress treatments, but there was no evidence of a condition-dependent decrease in eyespan under high stress. Similar but more complex patterns were observed for female eyespan, with evidence of additivity under low stress and heterosis under high stress. These results do not support the hypothesis that reduced sexual ornament size in meiotic drive males is due to a condition-dependent response to the putative increase in mutation load. Instead, reduced eyespan likely reflects compensatory resource allocation to different traits in response to drive-mediated destruction of sperm.

AbstractThe amount of genetic variation for fitness within populations tends to exceed that expected under mutation-selection-drift balance. Several mechanisms have been proposed to actively maintain polymorphism and account for this discrepancy, including antagonistic pleiotropy (AP), where allelic variants have opposing effects on different components of fitness. Here we identify a non-coding indel polymorphism in thefruitlessgene ofDrosophila melanogasterand measure survival and reproductive components of fitness in males and females of replicate lines carrying one or the other allele. Expressing the fruitless region in a hemizygous state we observe a pattern of AP, with one allele resulting in greater reproductive fitness while the other confers greater survival to adulthood. Different fitness effects were observed in an alternative genetic background, suggesting widespread epistatic effects. Our findings link sequence-level variation at a single locus with complex effects on a range of fitness components, thus helping to explain the maintenance of genetic variation for fitness. Transcription factors, such asfruitless, may be prime candidates for targets of balancing selection since they interact with multiple target loci and their associated phenotypic effects.

The maternal inheritance of mitochondrial genomes entails a sex-specific selective sieve, whereby mutations in mitochondrial DNA can only respond to selection acting directly on females. In theory, this enables male-harming mutations to accumulate in mitochondrial genomes if they are neutral, beneficial, or only slightly deleterious to females. Ultimately, this bias could drive the evolution of male-specific mitochondrial mutation loads, an idea known as mother’s curse. Earlier work on this hypothesis has mainly used small Drosophila panels, in which naturally-sourced mitochondrial genomes were coupled to an isogenic nuclear background. However, the lack of nuclear genetic variation has precluded robust generalization. Here we test the predictions of mother’s curse using a large Drosophila mito-nuclear genetic panel, comprising 9 isogenic nuclear genomes coupled to 9 mitochondrial haplotypes, giving a total of 81 different mito-nuclear genotypes. This enables systematic testing of both mito-nuclear interactions and mitochondrial genetic variance. Following a predictive framework, we performed a screen for wing centroid size, as this trait is highly sexually dimorphic and depends on metabolic function. We confirmed that the trait is sexually dimorphic, and show high levels of mito-nuclear epistasis. Importantly, we report that mitochondrial genetic variance has a greater impact on male versus female Drosophila , in 8 out of the 9 nuclear genetic backgrounds. These results demonstrate that the maternal inheritance of mitochondrial DNA does indeed modulate male life-history traits in a more generalisable way than previously envisaged. ### Competing Interest Statement The authors have declared no competing interest.