Inhibitors In Preclinical Models Research Articles

Abstract C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C026.

Hyaluronidase improves the efficacy of nab-paclitaxel after prolonged angiogenesis inhibition in preclinical models for esophagogastric cancer

BackgroundLong-term anti-angiogenesis leads to pruned vasculature, densely deposited extracellular matrix (ECM), and consequently reduced chemotherapy delivery in esophagogastric cancer (EGC). To address this issue, we evaluated the efficacy of adding a hyaluronidase or a NO-donor to the regimen of chemotherapy and anti-angiogenic drugs. MethodsA patient-derived EGC xenograft model was developed. Grafted mice were randomly assigned to four experimental groups and one control group. The experimental groups received DC101, a murine angiogenesis inhibitor, and nab-paclitaxel (NPTX), with the addition of hyaluronidase (PEGPH20), or NO-donor (nitroglycerine, NTG), or their combination, respectively. We compared tumor growth during 17 days of treatment. We performed immunohistochemistry for ECM components hyaluronan (HA) and collagen, CD31 for endothelial cells, and γH2AX for DNA damage. The positively stained areas were quantified, and vessel diameters were measured using QuPath software. ResultsProlonged DC101 treatment induced deposition of HA (p<0.01) and collagen (p<0.01). HA was effectively degraded by PEGPH20 (p<0.001), but not by NTG as expected. Both PEGPH20 (p<0.05) and NTG (p<0.01) dilated vessels collapsed in response to long-term DC101 treatment. However, only PEGPH20 (rather than NTG) was found to significantly inhibit tumor growth (p<0.05) in combination with NPTX and DC101. ConclusionsThese findings suggest that the mechanical barrier of HA is the major reason responsible for the resistance developed during prolonged anti-angiogenesis in EGC. Incorporating PEGPH20 into the existing treatment regimen is promising to improve outcomes for patients with EGC.

JNJ-77242113, a highly potent, selective peptide targeting the IL-23 receptor, provides robust IL-23 pathway inhibition upon oral dosing in rats and humans

The interleukin (IL)-23 pathway is a pathogenic driver in psoriasis, psoriatic arthritis, and inflammatory bowel disease. Currently, no oral therapeutics selectively target this pathway. JNJ-77242113 is a peptide targeting the IL-23 receptor with high affinity (KD: 7.1 pM). In human cells, JNJ-77242113 potently and selectively inhibited proximal IL-23 signaling (IC50: 5.6 pM) without impacting IL-12 signaling. JNJ-77242113 inhibited IL-23–induced interferon (IFN)γ production in NK cells, and in blood from healthy donors and psoriasis patients (IC50: 18.4, 11 and 9 pM, respectively). In a rat trinitrobenzene sulfonic acid-induced colitis model, oral JNJ-77242113 attenuated disease parameters at doses ≥ 0.3 mg/kg/day. Pharmacologic activity beyond the gastrointestinal tract was also demonstrated. In blood from rats receiving oral JNJ-77242113, dose-dependent inhibition of ex vivo IL-23–stimulated IL-17A production was observed. In an IL-23–induced rat skin inflammation model, JNJ-77242113 inhibited IL-23–induced skin thickening and IL-17A, -17F and -22 gene induction. Oral dosing of JNJ-77242113 in healthy human volunteers inhibited ex vivo IL-23–stimulated IFNγ production in whole blood. Thus, JNJ-77242113 provided selective, systemic IL-23 pathway inhibition in preclinical models which translated to pharmacodynamic activity in healthy human volunteers, supporting the potential for JNJ-77242113 as a selective oral therapy for IL-23–driven immune-mediated diseases.

Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores antitumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show selective hypermethylation and silencing of the cytoplasmic double-stranded DNA (dsDNA) sensor CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase (TE-RT) activates cGAS, triggering viral mimicry and stimulating antitumor immunity. In summary, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous RT activity to the mechanism of action of a US Food and Drug Administration-approved oncology drug.

Enhancing Standard of Care Chemotherapy Efficacy Using DNA-Dependent Protein Kinase (DNA-PK) Inhibition in Preclinical Models of Ewing Sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Abstract 1924: RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical models

Abstract Background: KRASG12C-GDP Inhibitors such as sotorasib and adagrasib have demonstrated clinical benefit in lung cancer patients harboring an oncogenic KRASG12C mutation. However, the durability of monotherapy benefit is limited by the development of resistance. Hence, identification of novel treatment approaches to overcome resistance and extend duration of benefit becomes necessary to address this unmet clinical need. Experimental Design: We generated two NSCLC models of KRASG12C−GDP inhibitor acquired resistance, named sotorasib-R LU65 (cell-line derived, in vitro) and adagrasib-R LUN156 (PDX-derived, in vivo) following extended treatment with sotorasib and adagrasib, respectively. We then performed transcriptomic and mass spectrometry-based phosphoproteomics analyses on baseline and KRASG12C-GDP inhibitor-resistant samples to determine resistance and escape mechanisms. Results: In the sotorasib-R LU65 cells, we discovered upregulation of various RTKs including HER2, HER3, and AXL using a mass spectrometry-based proteomics approach and Immunoblot analysis. Simultaneously, from a transcriptome analysis, we observed activation of RAS and PI3K/AKT signaling in these cells. In the adagrasib-R LUN156 model, increased phosphorylation of MET was observed via phospho-RTK array and by immunoblotting. Transcriptomic analysis revealed an enrichment for RAS pathway dependency in the adagrasib-R tumor models. RAS GTP-binding assessed by RAF-RBD pulldown and multiple reactions monitoring mass-spectrometry suggest increased GTP-bound RAS isoforms in sotorasib-R LU65 cells and active RAS signaling in adagrasib-R LUN156. Given the convergence of our findings on re-activation/maintenance of wild-type RAS signaling as a means of resistance to KRASG12C-GDP-state inhibitors, we hypothesized that RMC-7977, a tri-complex RASMULTI-GTP inhibitor, which inhibits signaling via mutant and wild-type RAS, had the potential to counteract these mechanisms. We tested RMC-7977 monotherapy, which drove complete tumor regressions in the adagrasib-R LUN156 model. Additionally, RMC-7977 attenuated tumor growth in sotorasib-R cell derived LU65 xenograft model wherein we observed co-activation of PI3K/AKT signaling. Thus, we tested a combination with the pan-PI3K inhibitor pictilisib and observed combinatorial activity consistent with our findings. Conclusions: These data suggest that in KRASG12C-GDP inhibitor-resistant models RTK activation maintains MAPK dependency and in the case of one model, PI3K signaling. Consistent with these mechanisms, RMC-7977 as a single agent or in combination with pictilisib drove significant tumor regressions in these models. These preclinical results indicate RASMULTI-GTP inhibition alone or combination with PI3K inhibition has the potential to address KRASG12C-GDP inhibitor resistance. Citation Format: Hitendra S. Solanki, Harshit Shah, Denis Imbody, Bina Desai, Paul A. Stewart, Bin Fang, Yaakov Stern, Lancia N. Darville, John M. Koomen, Andriy Marusyk, Ethan Ahler, Ida Aronchik, Mallika Singh, Eric B. Haura. RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical 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 1924.

Abstract 7592: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers such as androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combination of CDK4/6, AR, and PI3K-AKT inhibitors in preclinical models of LAR TNBC. The MDAMB453 and MFM223 (AR positive/Rb intact/PIK3CAmutant) LAR TNBC cells were treated with each inhibitor alone or in combination. Drug sensitivity was assessed using colony formation, cell viability, and live-cell analysis. The Loewe additivity equation was used to measure the additive drug effects. Immunoblot analysis was used to evaluate cell cycle and PI3K-AKT signaling and ARE luciferase assay was used to assess AR transcriptional activity. MDAMB453 and MFM223 cells were sensitive to the single agents palbociclib, alpelisib, and capivasertib (IC50, ~500 nM), which inhibited CDK4/6, PI3Kα, and AKT, respectively. Enzalutamide, an AR antagonist, exerted no growth inhibitory activity (IC50, 15-25 μM). Treatment with palbociclib, alpelisib, or capivasertib did not alter the AR protein levels or transcriptional activity. The combination of palbociclib+capivasertib synergistically inhibited the proliferation of MDAMB453 and MFM223 cells (synergistic scores 7.34, p=5.81-11, and 4.78, p=0.012, respectively) better than palbociclib+alpelisib. Enzalutamide did not enhance either combination. Palbociclib+capivasertib inhibited PI3KCA-mutant and Rb-intact LAR TNBC PDX growth more potently than palbociclib+alpelisib (p=0.046). Treatment of LAR TNBC cells with palbociclib suppressed Rb phosphorylation and resulted in adaptive activation of P-AKTS473/T308 and the AKT substrates GSK3β, PRAS40, and FoxO3a after 24 h. Capivasertib inhibited the phosphorylation of AKT and AKT substrates, following CDK4/6 blockade with palbociclib, more potently than alpelisib. PI3Kβ inhibitors (TGX221, GSK2636771) did not block AKT substrate upregulation, suggesting PI3Kβ does not mediate adaptive responses. Rictor knockdown failed to reduce AKT activation following palbociclib treatment, suggesting an mTORC2-independent mechanism. Human Phospho-kinase Array of MDA-MB-453 cells treated with palbociclib showed time dependent upregulation of GSK3βS9, STAT3Y727 and PDFGRβY751. PDGFRβ activation after palbociclib treatment was confirmed in MFM223 cells. PDGFRβ siRNAs in MDAMB453 cells blocked P-AKTS473 upregulation after palbociclib treatment. Treatment with palbociclib and the PDGFRβ small-molecule antagonist CP673451 arrested LAR TNBC growth similar to palbociclib+capivasertib, suggesting an adaptive response involving PDGFRβ signaling. In conclusion, this study supports the clinical testing of the palbociclib and capivasertib combination for LAR TNBC patients and reveals adaptive responses after palbociclib treatment through PDGFRβ signaling. Citation Format: Rosario Chica-Parrado, Gun Min Kim, Yasuaki Uemoto, Dan Ye, Fabiana Napolitano, Chang-Ching Lin, Emmanuel Bikorimana, Kyung-min Lee, Saurabh Mendiratta, Ariella B. Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC) [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 7592.

Abstract 6578: N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity

Abstract Background. Neutrophil elastase (ELANE) - a neutrophil-derived serine protease - kills a wide range of cancer cells without harming non-cancer cells through a unique killing program involving histone H1 and the death domain of CD95. Although ELANE attenuates tumor growth and induces anti-tumor immunity following intratumoral delivery (Cui et al., Cell, 2021), systemic delivery is stymied by the presence of serine protease inhibitors in blood (eg. alpha-1-antitrypsin (A1AT) which rapidly inhibit its enzymatic activity essential for its anti-cancer function. To overcome this challenge, we developed N17465, an enzyme that resists serine protease inhibitors in blood, and tested its effects on tumor growth, both as a monotherapy and in combination with checkpoint inhibitors. Methods. Enzymatic properties: N17465 enzymatic activity was quantified in the +/- of A1AT or plasma in vitro, and in blood following intravenous (I.V) injection into tumor-bearing mice. Enzyme activity assays included a fluorogenic substrate AAPV-AMC and the C-terminal domain of CD95 (amino acids212-335). Anti-cancer properties: For in vitro studies, cells were treated with N17465 and viability was quantified by calcein-AM and immunogenic cell death (ICD) markers. Cancer/non-cancer cells included human/murine cell lines and primary cells from ovarian cancer (OvCa) patients. For in vivo studies, N17465 was injected I.V and effects on tumor growth, immunology and survival were assessed as monotherapy or in combination with a checkpoint inhibitor (anti-CTLA4) in pre-clinical models. Results. N17465 maintained enzymatic activity following incubation with A1AT or mouse/human plasma in vitro, and following I.V injection into CT26 tumor-bearing mice in vivo. Importantly, protecting N17465 from serine protease inhibitors did not interfere with its ability to cleave CD95 (therapeutic target) or selectively kill cancer cells in vitro. Indeed, N17465 induced ICD markers, killed a wide range of murine/human cancer cells and also well tolerated by non-cancer cells. Systemically delivered N17465 produced durable tumor regression in a murine CT26 colon cancer model resulting in ~50% tumor-free mice, induced a favorable immune profile, and synergized with anti-CTLA4. N17465 also exhibited efficacy across a range of xenograft models spanning human colon, prostate, and lung cancer, as well as OvCa patient-derived CDX models. Conclusions. Altogether, our data demonstrate that N17465 selectively kills cancer cells, produces durable responses in mice, induces favorable immunology and synergies with checkpoint inhibitors in pre-clinical models. Its ability to maintain enzymatic activity in blood following systemic delivery, selectively kill a wide range of cancer cells, and stimulate anti-tumor immunity warrants further investigation of this unique therapeutic modality across a wide range of solid tumors. Citation Format: Ravindra Gujar, Chang Cui, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Lev Becker. N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity [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 6578.

Abstract 7052: p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer

Abstract The use of mitochondrial inhibitors to target oxidative phosphorylation (OXPHOS) in cancer treatment presents a challenge due to dose-limiting toxicities. Moreover, while glycolysis-deficient cancers are vulnerable to OXPHOS inhibition in preclinical models, the full extent of phenotypical and mechanistic consequences of inhibiting OXPHOS in cancers capable of glycolysis is not yet well understood. Our results presented here offer promising insights into potential therapeutic gains from combining p53 restoration strategies with OXPHOS inhibitors, even when applied to glycolysis-competent CRC cells. Treatment with mitochondrial complex I inhibitors did not cause energy stress in CRC cells capable of glycolysis. It does, however, induce DNA replication stress, apparent through an observed cell cycle arrest at the S phase, enrichment of the G2/M DNA-damage checkpoint regulation pathway, and replication fork slowdown. Intriguingly, CRC cells harboring wildtype p53 exhibited more severe replication stress than those carrying mutant p53. Furthermore, siRNA knockdown of p53 attenuates replication stress and reduces cell cycle arrest, underlining the important role of p53 in CRC cell responses to OXPHOS inhibition. Our targeted metabolomics analysis reveals that OXPHOS inhibition results in reductions in the purine nucleotides, adenine monophosphate (AMP) and guanine monophosphate (GMP), as well as the pyrimidine nucleotide, uridine monophosphate (UMP), in CRC cells, regardless of their p53 mutational status. By supplementing cell culture mediums with the purine nucleobases adenine and guanine, and the pyrimidine nucleoside uridine, we observed a partial reversal of the replication fork slowdown and reductions in cell viability. This suggests nucleotide deficiencies are involved in the induction of DNA replication stress caused by OXPHOS inhibition. The nucleotide deficiencies were associated with a decrease in the nucleobase precursor aspartate. By adding aspartate at a supraphysiological concentration, to overcome the low expression of the excitatory amino acid transporter 1 required for cellular import of aspartate, we were able to restore the levels of nucleotides. Collectively, our findings suggest broader potential cancer treatment paradigms via OXPHOS targeting, extending beyond glycolysis-deficient cancers. Our data uncovers that CRC cells, which commonly exhibit the glycolytic phenotype, are susceptible to OXPHOS inhibition, with those carrying wildtype p53 showing heightened sensitivity. Therefore, p53 status could serve as a biomarker for predicting CRC responses to OXPHOS inhibitors. Moreover, our findings suggest that combined strategies of restoring p53 function, using small molecules such as APR-246, might enable reduced dosage of OXPHOS inhibitors in CRC treatment, thereby mitigating their dose-limiting toxicities. Citation Format: Xiao Hong Zhao, Man Man Han, Qian Qian Yan, Yi Meng Yue, Kai Hong Ye, Yuan Yuan Zhang, Liu Teng, Liang Xu, Xiao Jing Shi, Ting La, Yu Chen Feng, Ran Xu, Vinod K. Narayana, David P. De Souza, Tao Liu, Mark Baker, Rick F. Thorne, Xu Dong Zhang, Song Chen, Lei Jin. p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer [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 7052.

SHP2 inhibition displays efficacy as a monotherapy and in combination with JAK2 inhibition in preclinical models of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, are clonal hematopoietic neoplasms driven by mutationally activated signaling by the JAK2 tyrosine kinase. Although JAK2 inhibitors can improve MPN patients' quality of life, they do not induce complete remission as disease-driving cells persistently survive therapy. ERK activation has been highlighted as contributing to JAK2 inhibitor persistent cell survival. As ERK is a component of signaling by activated RAS proteins and by JAK2 activation, we sought to inhibit RAS activation to enhance responses to JAK2 inhibition in preclinical MPN models. We found the SHP2 inhibitor RMC-4550 significantly enhanced growth inhibition of MPN cell lines in combination with the JAK2 inhibitor ruxolitinib, effectively preventing ruxolitinib persistent growth, and the growth and viability of established ruxolitinib persistent cells remained sensitive to SHP2 inhibition. Both SHP2 and JAK2 inhibition diminished cellular RAS-GTP levels, and their concomitant inhibition enhanced ERK inactivation and increased apoptosis. Inhibition of SHP2 inhibited the neoplastic growth of MPN patient hematopoietic progenitor cells and exhibited synergy with ruxolitinib. RMC-4550 antagonized MPN phenotypes and increased survival of an MPN mouse model driven by MPL-W515L. The combination of RMC-4550 and ruxolitinib, which was safe and tolerated in healthy mice, further inhibited disease compared to ruxolitinib monotherapy, including extending survival. Given SHP2 inhibitors are undergoing clinical evaluation in patients with solid tumors, our preclinical findings suggest that SHP2 is a candidate therapeutic target with potential for rapid translation to clinical assessment to improve current targeted therapies for MPN patients.

Insights into how adeno-squamous transition drives KRAS inhibitor resistance

The histologic transformation of adenocarcinoma (ADC) to squamous cell carcinoma (SCC), known as adeno-squamous transition or AST, is frequently observed in patients with lung cancer undergoing cancer therapy. In this issue, Tong and colleagues investigate genetic and epigenetic mechanisms that drive AST to confer resistance to KRAS inhibitors in preclinical models and patients.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Abstract C047: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of KRAS signaling through targeting the RAF-MEK-ERK kinase cascade resulted in further reliance on autophagy. We found that targeting this increased reliance on autophagy with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) together with MEK or ERK inhibition (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen in PDAC cells to identify other targetable mediators of autophagy. We identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition by the clinical candidate inhibitor apilimod resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition induced intracellular vacuolization. These PIKfyve-regulated vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, PIKfyve inhibition prevented the increased autophagic flux we observed when we inhibited MEK with the clinical stage MEKi, mirdametinib. As a result, co-targeting MEK and PIKfyve led to synergistic impairment of PDAC cell proliferation. Similar results were observed following dual inhibition of KRAS and PIKfyve. We found the synergistic growth inhibition was due in part to a substantial induction of apoptosis unique to combination treatment. We then tested the combination of MEKi and PIKfyvei in a panel of patient derived PDAC organoids and observed a robust synergistic reduction in viability, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. Future work includes in vivo studies, currently underway, to determine the efficacy of single agent PIKfyve inhibition, as well as efficacy studies assessing combined MEKi and PIKfyvei in orthotopic mouse models of PDAC. Taken together, these findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of pancreatic cancer. Citation Format: Jonathan M. DeLiberty, Mallory K. Roach, Noah L. Pieper, Kristina Drizyte-Miller, Elyse G. Schechter, Runying Yang, Channing J. Der, Adrienne D. Cox, Clint A. Stalnecker, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C047.

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML

AbstractSmall molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)–rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

Abstract C104: Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC

Abstract Aberrant expression of the transcription factor MYC is found in most cancers including non-small cell lung cancer (NSCLC) and has critical roles in a wide range of oncogenic processes including immune evasion and EGFR inhibitor resistance. Despite MYC’s therapeutic attractiveness, potent inhibitors of MYC activity have been elusive, due to its intrinsically disordered protein structure and tightly autoregulated expression. We have developed NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC Omega epigenomic controller (NSCLC MYC-OEC) that is designed to target the insulated genomic domain of MYC. We have previously shown that NSCLC MYC-OEC effectively downregulates MYC expression pre-transcriptionally and have demonstrated that NSCLC MYC-OEC inhibits NSCLC patient-derived organoid viability in vitro and abrogates xenograft tumor growth in vivo. To identify combination treatments with NSCLC MYC-OEC, we retrospectively evaluated baseline MYC expression and real-world progression-free survival (PFS) in a cohort of 283 NSCLC patients treated with pembrolizumab, an anti-PD1 monoclonal antibody. This analysis revealed that NSCLC patients with MYC-overexpressing tumors exhibited shorter PFS when treated with pembrolizumab. Moreover, analysis of PD1 or PD-L1 transcript levels in tumors from 13,230 lung adenocarcinoma patients revealed a significant positive correlation between PD1 or PD-L1 and MYC mRNA levels, consistent with a role for MYC in regulation of the immune response. In vivo evaluation of NSCLC MYC-OEC activity and immune checkpoint immunotherapy (ICI) treatment of preclinical Lewis lung carcinoma syngeneic tumor models demonstrated that the combination of NSCLC MYC-OEC with anti-PD-L1 antibody enhanced inhibition of tumor growth and prolonged survival over administration when compared to either NSCLC MYC-OEC or anti-PD-L1 antibody monotherapy. Retrospective analysis of a cohort of 301 NSCLC patients also showed that patients with MYC-overexpressing tumors exhibited significantly shorter PFS when treated with osimertinib, a third generation EGFR inhibitor. To further understand this, we investigated the effects of NSCLC MYC-OEC and osimertinib treatment alone or in combination in EGFR mutant cells, H1975 and PC9. While NSCLC MYC-OEC and osimertinib monotherapy treatments resulted in downregulation of MYC protein expression in both cell lines, combination treatment led to a significantly greater decrease in MYC expression. Furthermore, the combination synergistically reduced H1975 and PC9 cell viability in vitro and led to a significantly greater inhibition of H1975 xenograft growth relative to each single agent in vivo. Together, these data provide preclinical proof-of-concept in NSCLC for development of OEC-mediated pre-transcriptional epigenomic inhibition of MYC expression in combination with immune checkpoint and EGFR inhibitors. Furthermore, these findings suggest that NSCLC MYC-OEC combination with ICI or EGFR-targeted therapy may improve patient outcomes. Citation Format: Defne Yarar, Eugine Lee, Padraich Flahardy, Cameron Vergato, Graeme Hodgson, Thomas McCauley. Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC [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 C104.

Abstract A117: Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines

Abstract Background: The PI3K-AKT-mTOR pathway is frequently activated in many cancer types and plays a central role in tumorigenesis. However, clinical use of therapies targeting this pathway is limited by compensatory vertical and horizontal feedback activation loops, which limit antitumor efficacy and lead to drug resistance. Combination therapy strategies employing simultaneous inhibition of multiple PI3K-AKT-mTOR pathway elements may overcome these resistance mechanisms and improve antitumor activity. nab-Sirolimus is a novel albumin-bound nanoparticle form of the mTOR inhibitor sirolimus that has significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects compared with conventional first generation mTOR inhibitors in preclinical models. Here, we analyzed the effects of nab-sirolimus combinations in PI3K-mutant breast cancer (BrCa) cells. Methods: PI3K mutated BrCa cell lines (MDA-MD-453, hormone receptor [HR]-; and MDA-MB-361, HR+) were incubated for 5 days with increasing concentrations of nab-sirolimus, the PI3K pathway inhibitors gedatolisib and alpelisib, or the AKT inhibitors miransertinib and capivasertib. The antiproliferative and cytotoxic effects of single agent and combination treatment were assessed using cell viability and cell death assays. Cell signaling was analyzed by western blot. Results: The antiproliferative effects of gedatolisib at low doses (2.5-10 nM) were enhanced by 61-71% in combination with nab-sirolimus (20 or 80 nM) in MDA-MB-453 cells. Limited cell death (4.2-9.6%) was observed in HR- MDA-MB-453 cells with low doses (2.5, 5, and 10 nM) of gedatolisib alone. In contrast, and despite the well-established cytostatic action of mTOR inhibitors, the addition of 20 and 80 nM nab-sirolimus led to high levels of cell death (up to 48.9%). Similar results were observed with alpelisib at 1 µM in combination with nab-sirolimus (20 or 80 nM), and also with capivasertib (1 µM) or miransertib (250 nM) combined with 80 nM nab-sirolimus. Western blot analysis demonstrated that gedatolisib or alpelisib alone each triggered upregulation of p4EBP1 whereas nab-sirolimus treatment alone activated a negative feedback loop through increased pAKT. Importantly, these compensatory feedback activation loops were both reversed by the combination of PI3K inhibitors and nab-sirolimus. Similar cell death and cell viability results were observed with nab-sirolimus plus PI3K inhibitors (gedatolisib or alpelisib) and AKT inhibitors (capivasertib or miransertib) in HR+ MDA-MB-361 cells. Conclusions: The antitumor effects of PI3K and AKT inhibitors were enhanced by the addition of the novel mTOR inhibitor nab-sirolimus. The improved effectiveness of the PI3K and mTOR inhibitor combination was due to the reciprocal overcoming of resistance mechanisms induced by single agent treatment. These data support a vertical PI3K pathway inhibition strategy using nab-sirolimus and PI3K/AKT inhibitors in PIK3CA-mutated BrCa, regardless of HR status. Citation Format: Sean Wallace, Khine Nyein Myint, Shihe Hou, Maria Zalath, Andrew Kwon, Brian McMorran, Igor Vivanco. Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines [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 A117.

Abstract C020: First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid tumors

Abstract Activation of the mTOR pathway plays a role in the oncogenesis and progression of many cancer types, and is frequently co-altered in RAS-driven tumors. First-generation mTORC1-selective inhibitors were limited by incomplete inhibition of 4EBP1 phosphorylation, while second-generation pan-mTOR inhibitors had a low therapeutic index due to mTORC2-mediated toxicity such as hyperglycemia. RMC-5552 is a first-in-class third-generation bi-steric mTOR inhibitor that is mTORC1-selective via its FKBP12-binding moiety and potently inhibits phosphorylation of the 4EBP1 tumor suppressor via a linked ATP-competitive moiety, addressing these key limitations of prior mTOR inhibitors in preclinical models. RMC-5552 is being investigated in a Phase I/Ib study (NCT04774952). Eligible patients (pts) were ≥18 yrs old with ECOG 0-1 and had advanced solid tumors that had failed or were ineligible for standard-of-care treatments. Pts received RMC-5552 as a weekly infusion. To address mucositis, a common toxicity in pts treated with mTOR inhibitors, pts prophylactically used dexamethasone mouthwash either alone or with tacrolimus mouthwash (TM), which binds to the RMC-5552 presenter protein FKBP12. Objectives included assessment of safety/tolerability (CTCAEv5), defining the maximum tolerated and recommended Phase 2 doses, pharmacokinetics, and preliminary efficacy (RECIST v1.1). As of June 12, 2023, 47 pts have been evaluated at 6 dose levels from 1.6 mg to 12 mg. The most common (&amp;gt; 20%) treatment-related adverse events (TRAE, any grade) were stomatitis/mucositis (45%), fatigue (40%), nausea (38%), decreased appetite (32%), and vomiting (23%). Treatment-related events of hyperglycemia occurred in 4% of pts (both grade 2) and were not dose limiting. The most common grade 3 TRAE at dose levels ≥6 mg was mucositis/stomatitis, which was dose-dependent and limited tolerability in the absence of TM. Between 8 and 12 mg, the rate of treatment related stomatitis/mucositis was 65% (15% grade 3) in pts treated without TM (N = 20) versus 30% (no grade 3) in pts treated with TM (N = 10). TM prophylaxis enabled dose escalation beyond dose levels previously considered intolerable due to mucositis (10-12 mg; escalation ongoing), and unbound plasma exposures at 8-12 mg were within the range that induced significant anti-tumor activity in preclinical models. The disease control rate was 67% among efficacy evaluable pts (N = 39). One pt with a salivary gland tumor harboring a PTEN mutation had a confirmed partial response (PR) with 78% reduction and 16 months duration of response. In addition, molecular responses of circulating tumor DNA were observed in pts with mTOR pathway variants. Updated efficacy results will be presented. These early data indicate that RMC-5552 monotherapy is tolerated at doses predicted to be efficacious, and that selective inhibition of mTORC1 alleviates mTORC2-mediated toxicity. Dose escalation is ongoing, and mucositis prophylaxis has enabled further dose intensification for future combination studies with RAS(ON) inhibitors. Citation Format: Alison M Schram, Abdul Rafeh Naqash, Eric B Haura, Jonathan W Riess, Susanna V Ulahannan, Sai-Hong I Ou, Anna Capasso, Pamela N Munster, Michael L Cheng, W Clay Gustafson, Bojena Bitman, Sumit Kar, Zhican Wang, Lin Tao, Justin G Meyerowitz, Howard A Burris. First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid 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 C020.

EPCO-45. CELLULAR HIERARCHIES OF ETMR ARE SHAPED BY ONCOGENIC MICRORNAS AND RECEPTOR-LIGAND INTERACTIONS

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive brain tumor that predominantly occurs in children under the age of four years. Most children succumb within 10 months of diagnosis, typically due to invasive tumor growth and therapy-refractory behavior. 95% of ETMR harbor activating genetic alterations of the chromosome 19 microRNA cluster (C19MC). Despite the identification of this presumed oncogenic driving event, its biological consequences remain understudied and targeted therapy options are lacking. Here, we set out to further explore the intracellular heterogeneity of ETMR and shed light onto its oncogenic mechanisms with the goal to identify novel therapeutic targets for this dismal disease. We have compiled and analyzed a cohort of 11 ETMR tumors using single-cell RNA sequencing and multiplexed spatial imaging. We reveal a common, spatially distinct cellular hierarchy, closely resembling physiological brain development. This hierarchy spans a highly proliferative neural stem cell-like population that gives rise to more differentiated neuron-like cells. C19MC, predominantly expressed in the malignant stem-like population, controls a transcriptional network governing stemness and lineage commitment, as resolved by genome-wide analysis of microRNA-mRNA interactions. Targeting specific C19MC members by antisense oligonucleotides results in reduced cell proliferation, suggesting potent new avenues for future therapeutic approaches. Seeking more immediate therapeutic targets, we performed comprehensive cell-cell interaction analyses and reveal that FGF-FGFR and Delta-Notch interactions are important to maintain oncogenic signaling across malignant cell populations. These interactions were successfully targeted using small molecule inhibitors in preclinical models and in one ETMR patient. Taken together, we identify different malignant ETMR cell populations that follow a common developmental hierarchy, explore the regulatory reach of C19MC activation, and identify an oncogenic receptor-ligand interaction network. Targeting this interaction network opens a powerful new rationale for more effective ETMR therapies.

Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription.

MYC+ lymphomas are refractory to standard of care and novel treatments that downregulate MYC are needed. The utility of enitociclib, a selective CDK9 inhibitor in this patient population, is demonstrated in preclinical models and patients. Enitociclib inhibits RNA polymerase II function conferring a transcriptional shift and depletion of MYC and MCL1. Enitociclib intermittent dosing downregulates transcription factors including MYC, providing a therapeutic window for durable responses in patients with MYC+ lymphoma.