PI3K Inhibitor Research Articles

The Acetic Acid Produced by Lactobacillus Species Regulates Immune Function to Alleviate PEDV Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) infection results in significant mortality among newborn piglets, leading to substantial economic setbacks in the pig industry. Short-chain fatty acids (SCFA), the metabolites of intestinal probiotics, play pivotal roles in modulating intestinal function, enhancing the intestinal barrier, and bolstering immune responses through diverse mechanisms. The protective potential of Lactobacillus delbrueckii, Lactobacillus johnsonii, and Lactococcus lactis was first noted when administered to PEDV-infected piglets. Histological evaluations, combined with immunofluorescence studies, indicated that piglets receiving L. lactis displayed less intestinal damage, with diminished epithelial cell necrosis and milder injury levels. Differences in immunofluorescence intensity revealed a significant disparity in antigen content between the L. lactis and PEDV groups, suggesting that L. lactis might suppress PEDV replication, the intestine. We then assessed short-chain fatty acid content through targeted metabolomics, finding that acetate levels markedly varied from other groups. This protective impact was confirmed by administering acetate to PEDV-infected piglets. Data suggested that piglets receiving acetate exhibited resistance to PEDV. Flow cytometry analyses were conducted to evaluate the expression of innate and adaptive immune cells in piglets. Sodium acetate appeared to bolster innate immune defenses against PEDV, marked by elevated NK cell and macrophage counts in mesenteric lymph nodes, along with increased NK cells in the spleen and macrophages in the bloodstream. Acetic acid was also found to enhance the populations of CD8+ IFN-γ T cells in the blood, spleen, and mesenteric lymph, CD4+ IFN-γ T cells in mesenteric lymph nodes and spleen, and CD4+ IL-4+T cells in the bloodstream. Transcriptome analyses were carried out on the jejunal mucosa from piglets with PEDV-induced intestinal damage and from healthy counterparts with intact barriers. Through bioinformatics analysis, we pinpointed 189 significantly upregulated genes and 333 downregulated ones, with the PI3K-AKT, ECM-receptor interaction, and pancreatic secretion pathways being notably enriched. This transcriptomic evidence was further corroborated by western blot and qPCR. Short-chain fatty acids (SCFA) were found to modulate G protein-coupled receptor 41 (GPR41) and 43 (GPR43) in porcine intestinal epithelial cells (IPEC-J2). Post-acetic acid exposure, there was a notable upsurge in the ZO-1 barrier protein expression in IPEC-J2 compared to the unexposed control group (WT), while GPR43 knockdown inversely affected ZO-1 expression. Acetic acid amplified the concentrations of phosphorylated PI3K and AKT pivotal components of the PI3K/AKT pathway. Concurrently, the co-administration of AKT agonist SC79 and PI3K inhibitor LY294002 revealed acetic acid's role in augmenting ZO-1 expression via the P13K/AKT signaling pathway. This study demonstrates that acetic acid produced by Lactobacillus strains regulates intestinal barrier and immune functions to alleviate PEDV infection. These findings provide valuable insights for mitigating the impact of PEDV in the pig industry.

Nuciferine reduces vascular leakage and improves cardiac function in acute myocardial infarction by regulating the PI3K/AKT pathway

The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen–glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.

SIRT3 MEDIATES THE CARDIOPROTECTIVE EFFECT OF THERAPEUTIC HYPOTHERMIA AFTER CARDIAC ARREST AND RESUSCITATION BY RESTORING AUTOPHAGIC FLUX VIA THE PI3K/AKT/MTOR PATHWAY.

Background : Postresuscitation cardiac dysfunction is a significant contributor to early death following cardiopulmonary resuscitation (CPR). Therapeutic hypothermia (TH) mitigates myocardial dysfunction due to cardiac arrest (CA); however, the underlying mechanism remains unclear. Sirtuin 3 (Sirt3) was found to affect autophagic activity in recent research, motivating us to investigate its role in the cardioprotective effects of TH in the treatment of CA. Methods : Sprague-Dawley rats were used to establish an in vivo CA/CPR model and treated with a selective Sirt3 inhibitor or vehicle. Survival rate, myocardial function, autophagic flux, and Sirt3 expression and activity were evaluated. H9C2 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro . The cells were transfected with Sirt3-siRNA and treated with the autophagy inhibitor chloroquine or the PI3K inhibitor LY294002, and cell viability and autophagic flux were assessed. Results : Rats exhibited decreased survival and impaired cardiac function after CA/CPR, which were alleviated by TH. Mechanistically, TH restored Sirt3 expression and autophagic flux, which were impaired by CA/CPR. Sirt3 inactivation diminished the capacity of TH to restore autophagic flux and partially abolished the improvements in myocardial function and survival. An in vitro study further showed that TH-induced restoration of disrupted autophagic flux by OGD/R was attenuated by pretreatment with Sirt3-siRNA, and this attenuation was partially rescued by the inhibition of PI3K/Akt/mTOR signaling cascades. Conclusions : Sirt3 mediates the cardioprotective effect of TH by restoring autophagic flux via the PI3K/Akt/mTOR pathway. These findings suggest the potential of Sirt3 as a therapeutic target for CA.

ENDOGENOUS β 3 -ADRENERGIC RECEPTOR ACTIVATION ALLEVIATES SEPSIS-INDUCED CARDIOMYOCYTE APOPTOSIS VIA PI3K/AKT SIGNALING PATHWAY.

β 3 -adrenergic receptor (β 3 -AR) has been proposed as a new therapy for several myocardial diseases. However, the effect of β 3 -AR activation on sepsis-induced myocardial apoptosis is unclear. Here, we investigated the effect of β 3 -AR activation on the cardiomyocyte apoptosis and cardiac dysfunction in cecal ligation and puncture (CLP)-operated rats and lipopolysaccharide (LPS)-treated cardiomyocytes. We found that β 3 -AR existed both in adult rat ventricular myocytes (ARVMs) and H9c2 cells. The expression of β 3 -AR was upregulated in LPS-treated ARVMs and the heart of CLP rats. Pretreatment with β 3 -AR agonist, BRL37344, inhibited LPS-induced cardiomyocyte apoptosis and caspase-3, -8, and -9 activation in ARVMs. BRL37344 also reduced apoptosis and increased the protein levels of PI3K, p-Akt Ser473 and p-eNOS Ser1177 in LPS-treated H9c2 cells. Inhibition of PI3K using LY294002 abolished the inhibitory effect of BRL37344 on LPS-induced caspase-3, -8, and -9 activation in H9c2 cells. Furthermore, administration of β 3 -AR antagonist, SR59230A (5 mg/kg), significantly decreased the maximum rate of left ventricular pressure rise (+dP/dt) in CLP-induced septic rats. SR59230A not only increased myocardial apoptosis, reduced p-Akt Ser473 and Bcl-2 contents, but also increased mitochondrial Bax, cytoplasm cytochrome c, cleaved caspase-9, and cleaved caspase-3 levels of the myocardium in septic rats. These results suggest that endogenous β 3 -AR activation alleviates sepsis-induced cardiomyocyte apoptosis via PI3K/Akt signaling pathway and maintains intrinsic myocardial systolic function in sepsis.

RfxCas13d-mediated inhibition of Circ1647 alleviates renal fibrosis via PI3K/AKT signaling pathway

Background Circular RNAs (CircRNAs) have been shown to be involved in the development of chronic kidney disease (CKD). This study aimed to investigate the role of Circ1647 in renal fibrosis, which is a hallmark of CKD. Methods In this study, we established a unilateral ureteral obstruction (UUO) model and delivered Circ1647 RfxCas13d knockdown plasmid into renal parenchymal cells via retrograde injection through the ureter followed by electroporation. After that, the pathological changes were determined by Hematoxylin and Eosin. Meanwhile, Immunohistochemistry, qRT-PCR and Western blot were conducted to assess the degree of fibrosis. In addition, overexpressing of Circ1647 in renal tubular epithelial cells (TCMK1) was performed to investigate the underlying mechanisms of Circ1647. Results Our results displayed that electroporation-mediated knockdown of Circ1647 by RfxCas13d knockdown plasmid significantly inhibited renal fibrosis in UUO mice as evidenced by reduced expression of fibronectin and α-SMA (alpha-smooth muscle actin). Conversely, overexpression of Circ1647 in TCMK1 cells promoted the fibrosis. In terms of mechanism, Circ1647 may mediate the PI3K/AKT Signaling Pathway as demonstrated by the balance of the phosphorylation of PI3K and AKT in vivo and the aggravated phosphorylation of PI3K and AKT in vitro. These observations were corroborated by the effects of the PI3K inhibitor LY294002, which mitigated fibrosis post Circ1647 overexpression. Conclusion Our study suggests that Circ1647 plays a significant role in renal fibrosis by mediating the PI3K/AKT signaling pathway. RfxCas13d-mediated inhibition of Circ1647 may serve as a therapeutic target for renal fibrosis in CKD.

Chicoric acid advanced PAQR3 ubiquitination to ameliorate ferroptosis in diabetes nephropathy through the relieving of the interaction between PAQR3 and P110α pathway

ABSTRACT Purpose This study aimed to examine the impact of CA on DN and elucidate its underlying molecular mechanisms of inflammation. Methods We fed C57BL/6 mice injected with streptozotocin to induce diabetes. In addition, we stimulated NRK-52E cells with 20 mmol/L d-glucose to mimic the diabetic condition. Results Our findings demonstrated that CA effectively reduced blood glucose levels, and improved DN in mice models. Additionally, CA reduced kidney injury and inflammation in both mice models and in vitro models. CA decreased high glucose-induced ferroptosis of NRK-52E cells by inducing GSH/GPX4 axis. Conversely, the ferroptosis activator or the PI3K inhibitor reversed positive effects of CA on DN in both mice and in vitro models. CA suppressed PAQR3 expression in DN models to promote PI3K/AKT activity. The PAQR3 activator reduced the positive effects of CA on DN in vitro models. Moreover, CA directly targeted the PAQR3 protein to enhance the ubiquitination of the PAQR3 protein. Conclusion Overall, our study has uncovered that CA promotes the ubiquitination of PAQR3, leading to the attenuation of ferroptosis in DN. This effect is achieved through the activation of the PI3K/AKT signaling pathways by disrupting the interaction between PAQR3 and the P110α pathway. These findings highlight the potential of CA as a viable therapeutic option for the prevention of DN and other forms of diabetes.

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [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 534.

Abstract 3412: Benralizumab for treatment of eosinophil-related cutaneous adverse events associated with checkpoint inhibitors and anticancer targeted therapies: Phase 2 study

Abstract Background: Eosinophil-related cutaneous adverse events (ercAEs) are common toxicities of PI3K inhibitors, antibody drug conjugates (ADCs), checkpoint inhibitors (CPIs), and targeted cancer therapies. While systemic corticosteroids are effective, their use is limited by additional toxicities and high recurrence. Benralizumab is an anti-IL-5Rα monoclonal antibody that induces eosinophil depletion via antibody-dependent cellular cytotoxicity, and is FDA-approved for severe eosinophilic asthma but has not been studied for ercAEs. Methods: We conducted a single-center, single-arm, Phase 2 trial (NCT04552288) of benralizumab in cancer patients with Grade 2/3 ercAEs from PI3K inhibitors, ADCs, CPIs or targeted therapies. Patients received benralizumab 30 mg subcutaneously every 4 weeks for 3 doses, then every 8 weeks for 3 doses. The primary endpoint was clinical response, defined as reduction in ercAE Grade to 0/1 at Week 4. Secondary endpoints included changes in quality of life, rash body surface area (BSA), cytokines and eosinophil biomarkers, as well as adverse events (AEs). Results: Forty-seven patients were enrolled, predominantly with breast (55.3%), bladder (10.6%), melanoma (10.6%), or kidney (6.4%) cancer. Baseline ercAEs were Grade 2 (48.9%) and Grade 3 (51.1%), most often from PI3K inhibitors (46.8%), CPIs (21.3%), tyrosine kinase inhibitors (8.5%) or ADCs (8.5%). Overall, 32 of 42 evaluable patients (76.2%) had clinical response by Week 4, with decreased ercAE grade (mean 2.4 to 0.9, p<0.0001), improved quality of life, reduced rash BSA, decreased peripheral and skin eosinophils (all p<0.0001), and cytokine changes. Clinical response by Week 4 was seen in all patients with ercAEs from alpelisib (PI3K inhibitor; n=18) or enfortumab vedotin (ADC; n=4); p-value for both: p<0.05. Mild-to-moderate mucositis, diarrhea, and xerosis were the most common AEs, all unrelated to benralizumab. Conclusions: Benralizumab demonstrated favorable safety and efficacy for Grade 2/3 ercAEs from cancer therapies, especially alpelisib and enfortumab vedotin. Larger placebo-controlled trials are warranted to confirm these preliminary findings. Citation Format: Mario E. Lacouture, Tara Maier, Alexander Pan, Anna Chen, George Dranitsaris, Neil J. Shah, Sarat Chandarlapaty, Chau Dang, Devika Gajria, Allison Gordon, Neil Iyengar, Pedram Razavi, Mark Robson, Ezra Rosen, Serena Wong, Ucalene Harris, Kwami Ketosugbo, Cindy Bravo, Manu Jain, Alina Markova. Benralizumab for treatment of eosinophil-related cutaneous adverse events associated with checkpoint inhibitors and anticancer targeted therapies: Phase 2 study [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 3412.

Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [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 2089.

Abstract 3790: HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway

Abstract High-temperature requirement protease A3 (HtrA3) is known to contribute to the progression of various carcinomas through tissue-specific actions, yet its precise role in colorectal cancer (CRC) remains unclear. Our study revealed that HtrA3 is significantly upregulated in CRC at both mRNA and protein levels. TCGA database analysis showed that higher HtrA3 expression in colon adenocarcinoma is linked to poorer patient survival. Moreover, increased HtrA3 expression in HCT116 cells boosted their proliferation, migration, and invasion, while decreasing HtrA3 in SW480 cells had the reverse effect. In HCT116 cells with high HtrA3 levels, there was an increase in p-PI3K, p-AKT, and p-FOXO1, which reduced upon treatment with the PI3K inhibitor LY294002. Conversely, in SW480 cells with lowered HtrA3, these molecular levels decreased and rose again after treatment with the PI3K activator 740Y-P. HtrA3 also promoted cell proliferation, migration, and invasion in the presence of LY294002 and 740Y-P. In vivo experiments demonstrated that HtrA3 overexpression accelerated xenograft tumor growth. This study confirms that HtrA3’s overexpression in CRC cells promotes their proliferation, migration, and invasion by activating the PI3K-AKT-FOXO1 signaling pathway. Therefore, HtrA3 could be a significant factor in the advancement of CRC and may serve as a potential prognostic marker for guiding targeted CRC therapies. Citation Format: Jing Zheng, Xianhua Huang, Xian-e Peng, Yunli Wu. HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway [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 3790.

Abstract 5841: A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with a devastating 5-year survival rate of 6.8%. The current standard of care, consisting of surgery, radiation, and temozolomide (TMZ), has remained unchanged for over the past 15 years despite its limited efficacy and the serious therapy-related adverse events associated to this regimen. Although some frequent genetic alterations have been identified in GBM, no targeted therapies have shown efficacy in these patients to date. Among these, the phosphoinositide 3-kinase (PI3K) pathway is a signaling network that regulates cell growth, proliferation, migration, and invasion. Extensive molecular characterization of GBM has identified alterations in the components of the PI3K pathway in 80% of primary GBMs, including amplification of receptor tyrosine kinases (RTKs), activating mutations in the PI3K catalytic subunit (PIK3CA), loss-of-function mutations in the PI3K regulatory subunit (PIK3R1), and loss of the major tumor suppressor of the pathway, phosphatase and tensin homologue (PTEN). Despite the universal upregulation of PI3K pathway signaling associated with these genetic alterations, clinical trials investigating PI3K inhibitors in GBM have failed due to insufficient antitumor activity and dose-limiting toxicities. To elucidate mechanisms of resistance to PI3K inhibition in GBM, we used a genome-wide functional CRISPR-Cas9 knockout screen to identify genes that mediate resistance to the dual PI3K/mTOR inhibitor XL765 in PTEN-null SF295 GBM cells. We identified reactivation of the PI3K pathway and inhibition of autophagy as potential resistance mechanisms to dual PI3K/mTOR inhibition. Using orthogonal approaches, we will functionally validate our candidate hits through mechanistic studies, with patient-derived models, and with in vivo orthotopic xenografts. Our findings will elucidate novel mechanisms of resistance to PI3K/mTOR inhibition in GBM and will streamline rational drug combinations aimed to overcome resistance, thus eventually maximizing therapeutic responses to PI3K inhibitors in GBM. Citation Format: Annalisa V. Ferrotta, Arianna Izawa-Ishiguro, Meaghan Grogan, Ingo K. Mellinghoff. A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme [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 5841.

Abstract 7577: A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation

Abstract Cancer cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Uncontrolled cancer cell proliferation, as well as immune suppressive macrophage proliferation, contribute to tumor growth and spread. Effort to suppress tumor cell growth using Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth, but resistance develops due to upregulation of the alternative pathway. We identified a novel series of allosteric tyrosine kinase receptor inhibitors that induce synthetic lethality in AML, lung carcinoma, pancreatic carcinoma, and other tumor cells and in proliferating macrophages in vitro and in vivo by simultaneously inhibiting cMyc and Tor and activating the stress activated kinase JNK, leading to p53 mediated cell cycle arrest. Select kinase inhibitors with a unique positioning of a single side chain inhibited CDK8/19 and thereby Myc while stimulating phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting both tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Jason Rivera, Marc Paradise, Zihan Xu, Giuliana Mognol, Judith Varner. A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation [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 7577.

Abstract 1929: Acquired resistance to sotorasib in KRASG12C mutant NSCLC is vulnerable to PI3K-mTOR pathway inhibition and regulated by 4E-BP1

Abstract Up to 40% of NSCLC patients with KRASG12C mutation respond to sotorasib. However, acquired resistance (AR) develops rapidly in all patients. The majority of AR is associated with unknown mechanisms. Twelve KRASG12C mutant NSCLC PDXs were developed from patient samples and characterized with WES, RNAseq, and RPPA analyses. Three PDXs treated with sotorasib showed complete tumor regression, and one showed growth inhibition. PDXs, PDX derived organoids (PDXO), and cell line derived xenografts (CDX) were with AR to sotorasib were generated. TC303AR and TC314AR PDXs were generated by prolonged continuous in-vivo treatment with sotorasib. Two isogeneic AR PDXOs from TC303 and TC314 PDXs were generated through continuous incremental dosing of sotorasib in organoid cultures. Both TC303AR and TC314AR PDXOs showed >100-fold resistance than sensitive counterparts. Over 100-fold resistance were also measured in two sotorasib resistant isogenic H23AR and H358AR cell lines. Both resistant PDXOs and cell lines showed resistance to adagrasib, another selective, KRASG12C inhibitor. H358AR CDXs were generated through continuous in-vivo treatment with sotorasib and multiple passages of CDX tissues. WES was performed for AR PDXs, CDX, PDXOs and cell lines and all retained the KRASG12C mutation, and no additional KRAS mutations were found. We performed RNAseq, RPPA, and mass spectrometry (MS) on TC303AR and TC314AR PDXs. Heatmaps showed significant sets of differentially regulated RNA and protein in AR vs. parental. Enrichment analysis found a significant upregulation of MTORC1 signaling in AR PDXs. Proteins involved in PI3K-AKT-mTOR pathway were significantly upregulated in AR. PI3K and AKT expression were also increased in H23AR and H358AR. Inhibition of PI3K, AKT and mTOR by copanlisib, MK2206 and everolimus respectively was synergistic with sotorasib in AR cells and PDXOs, with copanlisib being the most effective. Copanlisib significantly inhibited colony formation in AR cells. Downstream molecules in PI3K-AKT-mTOR pathway showed marked downregulation of pAKT, pmTOR, p70S6, pS6, pGSK3b, and pPRAS40 by copanlisib. The downstream 4E-BP1 and p4E-BP1 were remarkably high in AR cells, which was downregulated by copanlisib. To verify the role of PI3K, PI3Kα CRISPR-Cas9 knock-out clones were generated in both H23AR and H358AR cells. Absence of p4E-BP1 was associated with restoration of sotorasib sensitivity while high levels of p4E-BP1 phosphorylated by an mTOR independent pathway restored sotorasib resistance in the knock-out clones. When copanlisib was combined with sotorasib in treating the resistant TC303AR, TC314AR PDXs, H358AR CDX and H23AR xenograft tumors, antitumor effects were observed in every model. Inhibition of the PI3K pathway at different nodes is a vulnerability in KRASG12C mutant NSCLC with sotorasib AR and p4E-BP1 is a mediator of sotorasib resistance Citation Format: Ismail M. Meraz, Shuhong Wu, Mourad Majidi, Chenghui Ren, Yi Xu, Feng Meng, Lihui Gao, Renduo Song, Ran Zhang, Bingliang Fang, Qi Wang, Yuanxin Xi, Jing Wang, Sung Yun Jung, Jack A. Roth. Acquired resistance to sotorasib in KRASG12C mutant NSCLC is vulnerable to PI3K-mTOR pathway inhibition and regulated by 4E-BP1 [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 1929.

Abstract 2102: Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis

Abstract The allosteric inhibitor rigosertib (an experimental therapeutic) was previously identified as an effective inducer of G2M arrest and apoptosis in squamous cell carcinoma (SCC) keratinocytes isolated from patients with the rare genetic disease recessive dystrophic epidermolysis bullosa (RDEB), both in vitro and in vivo. Early clinical trial data treating RDEB-associated SCC with rigosertib has shown remarkable efficacy in a sub-set of patients and understanding the mechanism of action of rigosertib in this lethal cancer would provide rationale for patient stratification. Multiple mechanisms of action were previously attributed to rigosertib, including polo-like kinase-1 (PLK1), PI3-kinase, activated RAS and microtubule polymerization inhibition. Our published data comparing PLK1 siRNA with PI3-Kinase inhibitors, MAPK inhibitors and microtubule disruption favored PLK1 as a mechanism of action without definitive conclusion. We therefore sought to determine the mechanism of action using transcriptomic profiling. Flow cytometry experiments identified three main categories of time to increase G2M in nine separate RDEB SCC keratinocyte populations in culture ranging from 1 to 6 hours after exposure to 1uM rigosertib. Bulk-RNA sequencing of representative populations from time to G2M groupings failed to identify consistent transcriptional change after exposure to rigosertib. However, single cell sequencing was able to identify increases in sub-populations of cells associated with G2M as well as transient emergence of unique populations that were specific to individual patient populations after exposure to rigosertib in culture. Of note a transient population of RDEB SCC keratinocytes only present after 1 hour of exposure to rigosertib was characterized by increased expression of the potassium regulator KCNRG which has previously been associated with apoptosis in chronic lymphocytic leukemia. Ongoing work is determining the role this gene may play in the mechanism of apoptosis induction in RDEB SCC keratinocytes after exposure to rigosertib. Citation Format: Andrew P. South, Suhao Han, Paolo Fortina. Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis [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 2102.

Abstract 1752: Comprehensive genomic profiling of advanced breast cancer subtypes: Insights from liquid biopsy analysis and implications for personalized therapies

Abstract Background: Breast cancer stands as the most prevalent malignancy affecting women’s health. The disease consists of three subtypes, including hormone receptor (HR) positive, HER2-positive, and triple-negative breast cancer (TNBC). In recent years, the advent of targeted therapies such as PI3K inhibitors has significantly improved the prognosis of breast cancer patients. However, the detection of relevant molecular markers, such as PIK3CA mutations, relies on tissue samples, which imposes limitations on the clinical application of these therapies. Consequently, this study presents a comprehensive analysis of genomic profiling that compares breast cancer patients across distinct pathological classifications, utilizing a liquid biopsy approach. Methods: In this retrospective study, 214 patients with advanced breast cancer were recruited. Plasma samples collected prior to first line treatment were analyzed using PredicineCARE, a targeted next-generation sequencing (NGS) liquid biopsy assay, to detect somatic alterations in ctDNA of blood, including single nucleotide variations (SNVs), gene fusions, and copy number variations (CNVs). Results: Based on the IHC classifications of tumor tissue, this cohort comprised 119 HR-positive, 61 HER2-positive, and 34 TNBC patients. The assay identified a total of 1456 mutations and 2357 gene copy number variants in plasma samples. Among these, the most frequently mutated genes and those with copy number variations (top 5) in HR-positive patients were TP53 (40%), PIK3CA (39%), ATM (22%), ERBB2 (19%), and FGFR1 (19%). In HER2-positive patients, they were TP53 (39%), PIK3CA (31%), ERBB2 (30%), NTRK1 (20%), and RAD50 (20%). For TNBC patients, the top genes were TP53 (59%), ERBB2 (44%), PIK3CA (35%), NTRK1 (35%), and BRCA2 (26%). Regarding the PIK3CA gene, the most prevalent mutation site was H1047, occurring in 12.61% of HR-positive patients, 14.75% of HER2-positive patients, and 11.76% of TNBC patients. Similarly, both E542 (4.20% in HR-positive, 3.28% in HER2-positive, and 2.94% in TNBC patients) and E545 (3.36% in HR-positive, 3.28% in HER2-positive, and 2.94% in TNBC patients) showed equivalent prevalence across the three classifications. These findings suggest the potential efficacy of PI3K inhibitors in various pathological types of breast cancer. Conclusions: This study offers insights into the genomic landscape of advanced breast cancer subtypes through liquid biopsy. The observed prevalence of PIK3CA mutations supports the potential efficacy of PI3K inhibitors across these diverse types of breast cancer, laying the groundwork for personalized therapeutic strategies and biomarker identification for prognosis and targeted therapies. Citation Format: Xiaoran Liu, Yaxin Liu, Cancan Jia, Yue Zhang, Feng Xie, Haoran Tang, Shidong Jia, Huiping Li. Comprehensive genomic profiling of advanced breast cancer subtypes: Insights from liquid biopsy analysis and implications for personalized therapies [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 1752.

Abstract 7140: A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents

Abstract Background: Plant-derived compounds, including flavonoids, can be potent anti-cancer agents, but low bioavailability, lack of specificity, and complexity in their synthesis can hamper their clinical development. Here, we present seven novel flavonoid derivatives, and test them for their anti-tumor activity in various models derived from hematological cancers and solid tumors. Methods: MTT assessed anti-proliferative activity after 72h of exposure to increasing concentrations of drugs. Tubulin Polymerization Assay was performed with 3-fold dilutions (15-0.02 μM), compared to paclitaxel (1-3 μM) or vinblastine (3 μM) as references. The OncolinesProfiler analysis was used to compare IC50 fingerprints determined on the Oncolines cell line panel to the IC50 fingerprints of 213 pre-profiled reference drugs. Results: SDN207, SND210, SND216, SND462, SND504, SND524, and SND562 were designed and synthesized using a flavonoid polyphenol scaffold selectively modified to markedly increase cytotoxicity towards cancer cells. All seven compounds showed dose-dependent anti-proliferative activity across 15 lymphoma cell lines derived from germinal center B-cell-like (GCB) diffuse large B cell lymphoma (DLBCL) (n=6), activated B cell-like (ABC) DLBCL (n=2), mantle cell lymphoma (n=4), and marginal zone lymphoma (MZL) (n=3). Activity in the nM range was seen with five compounds: SDN207 (median IC50 1.6 nM; 95%CI, 1.3-3.2), SND210 (1.4 nM; 95%CI, 0.9-3.3), SND216 (19.5 nM; 95%CI, 10-38 nM), SND562 (28.9 nM; 95%CI, 15.4-57.7), and SND524 (46.7 nM; 95%CI, 32-133.3). Both other compounds had a median IC50 of 2 μΜ (SND462, 95%CI, 1.1-4.5 μM; SND504, 95%CI, 0.5-3.9 μM). There were no differences based on lymphoma histology. The anti-tumor activity was maintained in MZL cell lines with acquired resistance to BTK and PI3K inhibitors (n=3). Six compounds were also assessed for their anti-tumor activity in models other than lymphomas (SDN207, n=31; SND210, n=20; SND462, n=4; SND504, n=4; SND524, n=9; SND562, n=35), confirming the higher activity for SDN207, SND210, SND562, and SND524. Regarding the mechanism of action, up to now, a tubulin polymerization assay was performed for three compounds. Microtubule depolymerization (SND207), and microtubule polymerization (SND524, SND562) were observed at concentrations as low as 20 nM. The OncolinesProfiler identified the tubulin-targeting agents vinblastine, vincristine, paclitaxel, and docetaxel among the top reference compounds most correlated with SND207 and SND210 (i.e., Pearson correlation > 0.5). Conclusions: Novel compounds developed using plant-derived skeletons have shown in vitro anti-tumor activity in lymphomas, leukemias, and solid tumors, including models of resistance to targeted agents. Microtubule targeting appeared as a possible mechanism of action for at least four of them. Citation Format: Alberto J. Arribas, Eleonora Cannas, Eugenio Gaudio, Paulina Biniecka, Dan Stoicescu, Francesco Bertoni. A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents [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 7140.

Abstract 5836: CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells

Abstract Background: Copanlisib, a PI3K inhibitor with a higher selectivity on PI3Kα and PI3Kδ, is currently being evaluated as a single agent and in combination for lymphoma and solid tumor patients. We and others reported the synergism achieved by adding the BCL2 inhibitor venetoclax, and the combination is in its early clinical assessment (NCT04939272, NCT04572763, NCT03886649). To explore genes affecting the response to the combination, we performed a genome-wide CRISPR Cas9 screen in a marginal zone B cell lymphoma (MZL) cell line under drug exposure. Methods: We generated, via lentiviral infection, a stable Cas9-expressing VL51 line, which was then infected (MOI 0.3) with the genome-wide Brunello guide RNA library, interrogating 19114 genes. After puromycin selection, 40M cells underwent DNA extraction (T0), 40M were exposed to DMSO ( control arm), 40M to copanlisib (0.5nM) + venetoclax (25nM) (experimental arm) for 2 weeks. DNA at T0 and extracted at 2 weeks underwent Illumina sequencing according to the Broad Institute Genetic Perturbation Platform protocol. MAGeCK-MLE was used to model sgRNA read counts and assess the sgRNAs statistical enrichment (P<0.05 FDR< 0.15), GSEA for genesets enrichments. Results: In the DMSO-treated cells, 160 genes had a positive β value (i.e., the sgRNAs knocking down the genes were positively enriched after 2 weeks), while 1152 had a negative β value (i.e., the sgRNAs knocking down the genes were negatively enriched after two weeks), for a total of 1152 differential enriched targets. In the cells exposed to the drug combination, 207 genes had positive and 1324 negative β, for a total of 1531 differentially enriched targets. Genes defined as essential, according to DepMap, represented 768/1152 (67%) and 843/1531 (55%) in control and experimental arms, respectively. The experimental arm did not massively differ from the control arm as shown by 815 genes (615, 74%, defined as essential by DepMap) overlapping between the 2 conditions and by highly significant enrichments at GSEA. A few genes were relevant only in VL51 exposed to copanlisib/venetoclax and not to DMSO. Selected hits were individually validated. Knocking down VKORC1L1, XCL1, RFK, MAP2K6, POC1A, and WTN5A improved the activity of the combination. WTN5A was also validated using recombinant protein (10 ng/mL), which reduced the effect of copanlisib/venetoclax. SPARCL1, MAPK4, ID2, and MSTN silencing reduced the activity of the combination. MSTN, coding for myostatin, was also validated by adding the recombinant protein (10 ng/mL), which increased the drugs anti-tumor activity. Conclusions: A genome-wide genetic screening identified genes modulating the lymphoma cells response to the copanlisib/venetoclax combination, providing novel therapeutic targets or biomarkers. Citation Format: Sara Napoli, Alberto J. Arribas, Eleonora Cannas, Federica Fuzio, Luciano Cascione, Andrea Rinaldi, Emanuele Zucca, Andrea Alimonti, Anastasios Stathis, Francesco Bertoni. CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells [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 5836.

Abstract 1230: MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance

Abstract Adaptive resistance mechanisms compromise the long-term effectiveness of kinase-targeted agents dictating the need for strategic combination approaches. MTX-531 was computationally designed to selectively target both PI3K and wild type EGFR. MTX-531 exhibits low nanomolar potency against the PI3K isoform family and EGFR with a high degree of specificity predicted by co-crystal structural analyses. Full kinome screening confirmed that MTX-531 is exquisitely selective for its intended targets. We evaluated the pharmacological profile of MTX-531, whereupon single agent treatment with this lead molecule elicited a high incidence of durable tumor regressions in a broad panel of PIK3CA mutant CDX and PDX squamous head and neck (HNSCC) models. The combination of MTX-531 with RAS pathway inhibitors, including agents directed against KRAS G12C, led to durable regressions of KRAS mutant colorectal and pancreatic xenografts, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to hyperglycemia and hyperinsulinemia commonly seen with predecessor pan PI3K inhibitors. MTX-531 acts as an agonist of PPARγ, a structural feature of the molecule that is thought to mitigate hyperglycemia induced by PI3K inhibition. This unique attribute of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors. To the best of our knowledge, MTX-531 is the first reported pan-PI3K inhibitor that does not lead to hyperglycemia. The pharmaceutical profile of MTX-531 includes favorable cross-species oral bioavailability, microsomal stability, and a facile 3 step chemical synthesis. MTX-531, which has the unique capability of concurrently and selectively inhibiting PI3K and EGFR, illustrates the power of rational computational drug design to target multiple adaptive resistance mechanisms in a single molecule. The versatility of MTX-531 in both the single agent and combination settings offers a breadth of development strategies that continue to evolve as new combination candidates become available. MTX-531 is currently undergoing advanced preclinical development for anticipated first in human clinical trials in late 2024. Citation Format: Christopher E. Whitehead, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Rachel A. Mumby, June Chung, Jinju Li, Nathaniel Osher, Oluwadara Coker, Michelle Norris, Scott Kopetz, Veera Baladandayuthapani, Melinda Hollingshead, Sharad Verma, Judith S. Sebolt-Leopold. MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance [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 1230.

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [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 4608.

Abstract 7196: Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines

Abstract Background: Endocrine therapy (ET) is the standard of care for hormone receptor positive (HR+) breast cancer (BC), but ET resistance develops in most patients (pts). One resistance mechanism involves cross talk between the PI3K-AKT-mTOR and estrogen receptor (ER) signaling pathways, resulting in increased ER signaling and tumor proliferation. This is mitigated by mTOR inhibition as demonstrated by improved outcomes with exemestane plus the mTOR inhibitor (mTORi) everolimus in pts with HR+ advanced BC. While PI3K inhibitors (PI3Ki [e.g. alpelisib]) are efficacious in pts with HR+, PIK3CA-mutated BC, resistance can occur due to acquired genomic alterations in the PI3K-AKT-mTOR pathway. Combination therapy that targets a critical downstream node, mTOR, could delay or overcome acquired resistance due to activation of upstream components of the PI3K-AKT-mTOR pathway. nab-Sirolimus is an intravenously administered, albumin-bound nanoparticle form of the mTORi sirolimus, with significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects than oral mTORis in nonclinical tumor models. We evaluated the antiproliferative and cytotoxic effects of nab-sirolimus in combination with ET or PI3Kis in BC cell lines. Methods: HR+, PI3K-mutated BC cell lines (MCF7 or MDA-MB-361) were incubated for 5 days with increasing concentrations of nab-sirolimus and the selective ER degrader fulvestrant. MDA-MB-361 cells or HR-negative (HR−), PI3K-mutated BC cells, MDA-MD-453, were incubated for 5 days with increasing concentrations of nab-sirolimus and PI3K-AKT-mTOR pathway inhibitors gedatolisib or alpelisib. Antiproliferative/cytotoxic effects of single-agent and combination treatment were assessed via an automated trypan blue exclusion assay. Results: Addition of 20 or 80 nM nab-sirolimus to fulvestrant (12.5-500 nM) markedly decreased cell viability and nearly doubled cell death in MCF7 and MDA-MB-361 cells. Antiproliferative effects of low-dose gedatolisib (2.5-10 nM) were enhanced by 61-71% when nab-sirolimus (20 or 80 nM) was added to MDA-MB-453 cells. While low doses (2.5, 5, and 10 nM) of gedatolisib alone led to limited cytotoxicity (4.2-9.6%), up to 48.9% of cell death occurred when 20 or 80 nM nab-sirolimus was added. Results were similar with alpelisib (1 uM) plus nab-sirolimus (20 or 80 nM). Western blot showed increased p4EBP1 in response to fulvestrant or PI3Ki treatment, which was reversed with the addition of nab-sirolimus. Conclusions: nab-Sirolimus enhanced the cytotoxic effects of fulvestrant in HR+ BC cells and of PI3Kis in HR+ and HR− BC cells. Addition of nab-sirolimus to ET or other PI3K-AKT-mTOR pathway inhibitors may overcome mechanisms of resistance; further exploration is warranted to elucidate clinical relevance. Citation Format: Khine Nyein Myint, Sean Wallace, Shihe Hou, Maria Zalath, Brian McMorran, Andrew Kwon, Igor Vivanco. Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines [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 7196.