mTOR Inhibitor Research Articles

Lysosomal degradation targets mutant calreticulin and the thrombopoietin receptor in myeloproliferative neoplasms

AbstractSomatic mutants of calreticulin (CRT) drive myeloproliferative neoplasms (MPNs) via binding to the thrombopoietin receptor (MPL) and aberrant activation of the JAK/STAT pathway. Compared with healthy donors, platelets from mutant CRT-expressing patients with MPN display low cell surface MPL. Additionally, coexpression of MPL with an MPN-linked CRT mutant (CRTDel52) reduces cell surface MPL, suggesting that CRTDel52 may induce MPL degradation. We show that lysosomal degradation is relevant to the turnover of CRTDel52 and MPL. Furthermore, CRTDel52 increases the lysosomal localization and degradation of MPL. Mammalian target of rapamycin (mTOR) inhibitors reduce cellular CRTDel52 and MPL, secreted CRTDel52 levels, and impair CRTDel52–mediated cell proliferation. mTOR inhibition also reduces colony formation and differentiation of CD34+ cells from patients with MPN but not from healthy donors. Together, these findings indicate that low-surface MPL is a biomarker of mutant CRT-mediated MPN and that induced degradation of CRTDel52 and MPL is an avenue for therapeutic intervention.

Improvement of diabetes-induced spinal cord axon injury with taurine via nerve growth factor-dependent Akt/mTOR pathway

Diabetic neuropathy (DN) is a common neurological complication caused by diabetes mellitus (DM). Axonal degeneration is generally accepted to be the major pathological change in peripheral DN. Taurine has been evidenced to be neuroprotective in various aspects, but its effect on spinal cord axon injury (SCAI) in DN remains barely reported. This study showed that taurine significantly ameliorated axonal damage of spinal cord (SC), based on morphological and functional analyses, in a rat model of DN induced by streptozotocin (STZ). Taurine was also found to induce neurite outgrowth in cultured cerebral cortex neurons with high glucose exposure. Moreover, taurine up-regulated the expression of nerve growth factor (NGF) and neurite outgrowth relative protein GAP-43 in rat DN model and cultured cortical neurons/VSC4.1 cells. Besides, taurine increased the activating phosphorylation signals of TrkA, Akt, and mTOR. Mechanistically, the neuroprotection by taurine was related to the NGF–pAKT–mTOR axis, because either NGF-neutralizing antibody or Akt or mTOR inhibitors was found to attenuate its beneficial effects. Together, our results demonstrated that taurine promotes spinal cord axon repair in a model of SCAI in STZ-induced diabetic rats, mechanistically associating with the NGF-dependent activation of Akt/mTOR pathway.

Free ribosomal proteins as culprits for nucleolar stress

Nucleolar stress has been consistently linked to age-related diseases. In this issue, Sirozh etal.1 find that the common molecular signature of nucleolar stress is the accumulation of free ribosomal proteins, which leads to premature aging in mice; however, it can be reversed by mTOR inhibition.

Therapeutic potential of targeting Nrf2 by panobinostat in pituitary neuroendocrine tumors

We aimed to identify the druggable cell-intrinsic vulnerabilities and target-based drug therapies for PitNETs using the high-throughput drug screening (HTS) and genomic sequencing methods. We examined 9 patient-derived PitNET primary cells in HTS. Based on the screening results, the potential target genes were analyzed with genomic sequencing from a total of 180 PitNETs. We identified and verified one of the most potentially effective drugs, which targeted the Histone deacetylases (HDACs) both in in vitro and in vivo PitNET models. Further RNA sequencing revealed underlying molecular mechanisms following treatment with the representative HDACs inhibitor, Panobinostat. The HTS generated a total of 20,736 single-agent dose responses which were enriched among multiple inhibitors for various oncogenic targets, including HDACs, PI3K, mTOR, and proteasome. Among these drugs, HDAC inhibitors (HDACIs) were, on average, the most potent drug class. Further studies using in vitro, in vivo, and isolated PitNET primary cell models validated HDACIs, especially Panobinostat, as a promising therapeutic agent. Transcriptional surveys revealed substantial alterations to the Nrf2 signaling following Panobinostat treatment. Moreover, Nrf2 is highly expressed in PitNETs. The combination of Panobinostat and Nrf2 inhibitor ML385 had a synergistic effect on PitNET suppression. The current study revealed a class of effective anti-PitNET drugs, HDACIs, based on the HTS and genomic sequencing. One of the representative compounds, Panobinostat, may be a potential drug for PitNET treatment via Nrf2-mediated redox modulation. Combination of Panobinostat and ML385 further enhance the effectiveness for PitNET treatment.

Dichloroacetate and Quercetin Prevent Cell Proliferation, Induce Cell Death and Slow Tumor Growth in a Mouse Model of HPV-Positive Head and Neck Cancer.

Elevated glucose uptake and production of lactate are common features of cancer cells. Among many tumor-promoting effects, lactate inhibits immune responses and is positively correlated with radioresistance. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that decreases lactate production. Quercetin is a flavonoid compound found in fruits and vegetables that inhibits glucose uptake and lactate export. We investigated the potential role and mechanisms of DCA, quercetin, and their combination, in the treatment of HPV-positive head and neck squamous cell carcinoma, an antigenic cancer subtype in need of efficacious adjuvant therapies. C57Bl/6-derived mouse oropharyngeal epithelial cells, a previously developed mouse model that was retrovirally transduced with HPV type-16 E6/E7 and activated Ras, were used to assess these compounds. Both DCA and quercetin inhibited colony formation and reduced cell viability, which were associated with mTOR inhibition and increased apoptosis through enhanced ROS production. DCA and quercetin reduced tumor growth and enhanced survival in immune-competent mice, correlating with decreased proliferation as well as decreased acidification of the tumor microenvironment and reduction of Foxp (+) Treg lymphocytes. Collectively, these data support the possible clinical application of DCA and quercetin as adjuvant therapies for head and neck cancer patients.

Clinical study reveals the efficacy of sirolimus in treating primary immune thrombocytopenia: findings from a single-center study.

Immune thrombocytopenia (ITP) is an autoimmune disease that arises because of self-destruction of circulating platelets. The mechanism remains complicated and lacks a standard clinical treatment. Current first-line and second-line medications for ITP have shown limited effectiveness, necessitating the exploration of new therapeutic options. Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that has been demonstrated to inhibit lymphocyte activity, indicating potential for SRL in the treatment of ITP. This study aimed to evaluate the clinical efficacy of sirolimus as a second-line drug in patients with ITP. The starting dose of sirolimus for adults ranged from 2 to 4 mg/day, with a maintenance dose of 1 to 2 mg/day. For children, the starting dose was 1-2 mg/day, with a maintenance dose of 0.5-1 mg/day. The dosage could be adjusted if needed to maintain a specific blood concentration of sirolimus, typically between 5 and 15 ng/ml, throughout the treatment period. After 3 months, the overall response rate was 60% (12/20), with 30% of patients (6/20) achieving a complete response (CR) and 30% (6/20) achieving a partial response (PR). The CR rate at 6 months remained consistent with the 3-month assessment. No major adverse events were reported, indicating that sirolimus was well tolerated and safe. Analysis of peripheral blood Treg cell percentages in both the control and ITP showed no significant difference before treatment. The percentage of Treg cells increased after treatment with sirolimus, suggesting that sirolimus increases Treg cells. These findings suggest that sirolimus serves as an effective second-line treatment option for ITP, demonstrating favorable clinical efficacy.

Abstract LT09: Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies

Abstract Objectives: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. Design: Integrative multi-omics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, and patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining, and single-cell spatial transcriptomics were used to explore immunogenicity of diverse CCA. Results: We identified 3 distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumor growth in in-vitro and in-vivo models. The first group (ESTRO), with mostly fluke-associated CCAs, displayed activation in estrogen signaling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intra-tumor differences in AA mutation load were correlated to intra-tumor variation of different immune cell populations. Conclusion: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumorigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits. Citation Format: Jing Han Hong, Chern Han Yong, Hong Lee Heng, Jason Yongsheng Chan, Mai Chan Lau, Jianfeng Chen, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Peiyong Guan, Pek Lim Chu, Sheng Rong Ng, Xiaosai Yao, Peili Wang, Rong Xiao, Xian Zeng, Yichen Sun, Wei Liu, Joanna Koh, Felicia Yu Ting Wee, Jeffrey Chun Tatt Lim, Cedric Chuan Young Ng, Xiu Yi Kwek, Poramate Klanrit, Yaojun Zhang, Jiaming Lai, David Wai Meng Tai, Chawalit Pairojkul, Simona Dima, Irinel Popescu, Sen-Yung Hsieh, Ming-Chin Yu, Joe Yeong, Sarinya Kongpetch, Apinya Jusakul, Watcharin Loilome, Patrick Tan, Jing Tan, Bin Tean Teh. Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT09.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

TFE3 -Rearranged PEComa/PEComa-like Neoplasms : Report of 25 New Cases Expanding the Clinicopathologic Spectrum and Highlighting its Association With Prior Exposure to Chemotherapy.

Since their original description as a distinctive neoplastic entity, ~50 TFE3 -rearranged perivascular epithelioid cell tumors (PEComas) have been reported. We herein report 25 new TFE3 -rearranged PEComas and review the published literature to further investigate their clinicopathologic spectrum. Notably, 5 of the 25 cases were associated with a prior history of chemotherapy treatment for cancer. This is in keeping with prior reports, based mainly on small case series, with overall 11% of TFE3 -rearranged PEComas being diagnosed postchemotherapy. The median age of our cohort was 38 years. Most neoplasms demonstrated characteristic features such as nested architecture, epithelioid cytology, HMB45 positive, and muscle marker negative immunophenotype. SFPQ was the most common TFE3 fusion partner present in half of the cases, followed by ASPSCR1 and NONO genes. Four of 7 cases in our cohort with meaningful follow-up presented with or developed systemic metastasis, while over half of the reported cases either recurred locally, metastasized, or caused patient death. Follow-up for the remaining cases was limited (median 18.5 months), suggesting that the prognosis may be worse. Size, mitotic activity, and necrosis were correlated with aggressive behavior. There is little evidence that treatment with MTOR inhibitors, which are beneficial against TSC -mutated PEComas, is effective against TFE3 -rearranged PEComas: only one of 6 reported cases demonstrated disease stabilization. As co-expression of melanocytic and muscle markers, a hallmark of conventional TSC -mutated PEComa is uncommon in the spectrum of TFE3 -rearranged PEComa, an alternative terminology may be more appropriate, such as " TFE3 -rearranged PEComa-like neoplasms," highlighting their distinctive morphologic features and therapeutic implications.

Chemically engineered mTOR-nanoparticle blockers enhance antitumour efficacy

Hepatocellular carcinoma (HCC) is a highly prevalent and deadly type of cancer, and although pharmacotherapy remains the cornerstone of treatment, therapeutic outcomes are often unsatisfactory. Pharmacological inhibition of mammalian target of rapamycin (mTOR) has been closely associated with HCC regression. Herein, we covalently conjugated AZD8055, a potent mTORC1/2 blocker, with a small panel of unsaturated fatty acids via a dynamically activating linkage to enable aqueous self-assembly of prodrug conjugates to form mTOR nanoblockers. Cell-based experiments were carried out to evaluate the effects of the nanoblocker against hepatocellular carcinoma (HCC) cells. The orthotopic and subcutaneous HCC mouse models were established to examine its antitumour activity. Among several fatty acids as promoieties, linoleic acid-conjugated self-assembling nanoblocker exhibited optimal size distribution and superior physiochemical properties. Compared with free agents, PEGylated AZD8055 nanoblocker (termed AZD NB) was pharmacokinetically optimized after intravenous administration. Invivo investigations confirmed that AZD NB significantly suppressed tumour outgrowth in subcutaneous HCCLM3 xenograft, Hepatoma-22, and orthotopic Hepa1-6 liver tumour models. Strikingly, treatment with AZD NB, but not free agent, increased intratumour infiltration of IFN-γ+CD8+ T cells and CD8+ memory T cells, suggesting a potential role of the mTOR nanoblocker to remodel the tumour microenvironment. Overall, a single conjugation with fatty acid transformed a hydrophobic mTOR blocker into a systemically injectable nanomedicine, representing a facile and generalizable strategy for improving the therapeutic index of mTOR inhibition-based cancer therapy. The mTOR inhibition by chemically engineered nanoblocker presented here had enhanced efficacy against tumours compared with the pristine drug and thus has the potential to improve the survival outcomes of patients with HCC. Additionally, this new nanosystem derived from co-assembling of small-molecule prodrug entities can serve as a delivery platform for the synergistic co-administration of distinct pharmaceutical agents. This work was supported by the National Natural Science Foundation of China (32171368,81721091), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c and JNL-2022010B), State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (zz202310), and Natural Science Foundation of Shandong Province (ZR2023ZD59).

Novel Potential Therapeutic Targets in Autosomal Dominant Polycystic Kidney Disease from the Perspective of Cell Polarity and Fibrosis.

Autosomal dominant polycystic kidney disease (ADPKD), a congenital genetic disorder, is a notable contributor to the prevalence of chronic kidney disease worldwide. Despite the absence of a complete cure, ongoing research aims for early diagnosis and treatment. Although agents such as tolvaptan and mTOR inhibitors have been utilized, their effectiveness in managing the disease during its initial phase has certain limitations. This review aimed to explore new targets for the early diagnosis and treatment of ADPKD, considering ongoing developments. We particularly focus on cell polarity, which is a key factor that influences the process and pace of cyst formation. In addition, we aimed to identify agents or treatments that can prevent or impede the progression of renal fibrosis, ultimately slowing its trajectory toward end-stage renal disease. Recent advances in slowing ADPKD progression have been examined, and potential therapeutic approaches targeting multiple pathways have been introduced. This comprehensive review discusses innovative strategies to address the challenges of ADPKD and provides valuable insights into potential avenues for its prevention and treatment.

In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV.

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Discovery of a Long Half-Life AURKA Inhibitor to Treat MYC-Amplified Solid Tumors as a Monotherapy and in Combination with Everolimus.

Aurora kinase inhibitors, such as alisertib, can destabilize MYC-family oncoproteins and have demonstrated compelling antitumor efficacy. In this study, we report 6K465, a novel pyrimidine-based Aurora A inhibitor, that reduces levels of c-MYC and N-MYC oncoproteins more potently than alisertib. In an analysis of the antiproliferative effect of 6K465, the sensitivities of small cell lung cancer (SCLC) and breast cancer cell lines to 6K465 were strongly associated with the protein levels of c-MYC and/or N-MYC. We also report DBPR728, an acyl-based prodrug of 6K465 bearing fewer hydrogen-bond donors, that exhibited 10-fold improved oral bioavailability. DBPR728 induced durable tumor regression of c-MYC- and/or N-MYC-overexpressing xenografts including SCLC, triple-negative breast cancer, hepatocellular carcinoma, and medulloblastoma using a 5-on-2-off or once-a-week dosing regimen on a 21-day cycle. A single oral dose of DBPR728 at 300 mg/kg induced c-MYC reduction and cell apoptosis in the tumor xenografts for more than 7 days. The inhibitory effect of DBPR728 at a reduced dosing frequency was attributed to its uniquely high tumor/plasma ratio (3.6-fold within 7 days) and the long tumor half-life of active moiety 6K465. Furthermore, DBPR728 was found to synergize with the mTOR inhibitor everolimus to suppress c-MYC- or N-MYC-driven SCLC. Collectively, these results suggest DBPR728 has the potential to treat cancers overexpressing c-MYC and/or N-MYC.

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Abstract CT087: A phase 1/2, open-label, randomized, dose-finding and dose expansion study of gedatolisib in combination with darolutamide in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Progression to metastatic castration-resistant prostate cancer (mCRPC) occurs in most patients treated with androgen receptor signaling inhibitors (ARSi) for advanced disease. Preclinical studies demonstrate that AR and PI3K - AKT - mTOR (PAM) pathways interact through reciprocal negative feedback, whereby inhibition of one pathway activates the other. Thus, combining a PAM inhibitor with an ARSi may deliver improved anti-cancer activity in patients with mCRPC. A Phase 2 trial in 129 patients with mCRPC who progressed on abiraterone demonstrated improved median radiographic progression-free survival (rPFS) when samotolisib, a dual PI3K-mTOR inhibitor, was added to enzalutamide. These results form the basis for this clinical trial of gedatolisib, a potent PAM inhibitor, in combination with darolutamide in men with mCRPC who have previously progressed on ARSi (NCT06190899). Methods: This open-label, multicenter, Phase 1/2 study will evaluate the safety and efficacy of gedatolisib in combination with darolutamide in men with mCRPC who have progressed on ARSi. In Phase 1, 36 patients will be randomized to one of two dose arms to evaluate dose limiting toxicities (DLTs) and determine the recommended Phase 2 dose (RP2D). Gedatolisib will be administered once weekly for 3-weeks-on/1-week-off: Arm 1 - 120 mg and Arm 2 - 180 mg, with darolutamide 600 mg orally administered twice daily. Arm 2 may be dose de-escalated depending on the number of DLTs observed. In Phase 2, 12 additional patients will be enrolled at the RP2D (n= 30). Key inclusion criteria include adult males (≥ 18 years) with mCRPC who have progressed on or after treatment with one next-generation ARSi. Key exclusion criteria include males with adenocarcinoma with a small cell component and with ≥10% neuroendocrine type cells; prior treatment with PI3K, AKT, or mTOR inhibitor; prior chemotherapy or radiopharmaceutical therapy for mCRPC; uncontrolled type 1/2 diabetes; or active brain or leptomeningeal metastases. Primary endpoints for Phase 1 are safety and tolerability (incidence of DLTs, adverse events, and determination of maximum tolerated dose) determination of the recommended Phase 2 dose (RP2D), PK, and Bayesian Optimal Interval utility score. Primary endpoints for Phase 2 are rPFS rate at 6 months based on Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 with modifications as specified in Prostate Cancer Working Group 3 criteria. Secondary endpoints include rPFS rates at 9 and 12 months, overall rPFS, prostate-specific antigen response of ≥50% decrease from baseline at 4, 8, 12, and 16 weeks, overall response rate, duration of response, clinical benefit rate, overall survival rate at 18 and 24 months, and safety. The trial is currently open for enrollment. Citation Format: Adam Sharp, Alice Bernard-Tessier, Delphine Borchiellini, Johann de Bono, Karim Fizazi, Gwenaelle Gravis, David Lorente, Hakim Mahammedi, Begona Mellado, David Olmos, Simon Pacey, Jose Piulats, Sara P. Ramirez, Igor Gorbatchevsky, Elisabeth Heath. A phase 1/2, open-label, randomized, dose-finding and dose expansion study of gedatolisib in combination with darolutamide in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT087.

Abstract LB450: Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models

Abstract Capivasertib, a pan-AKT inhibitor, has recently been approved in combination with fulvestrant to treat ER+ HER2- breast cancer patients with one or more of PI3KCA/AKT1/PTEN alterations. However, as with many targeted therapies acquired drug resistance remains a challenge. In this study, we investigated the mechanisms of acquired capivasertib resistance in AKT1 mutant breast cancer models. We established two estrogen receptor positive (ER+) AKT1 E17K mutant breast cancer patient derived organoids (PDOs), that were sensitive to capivasertib in vitro. PDOs were chronically exposed to 1µM capivasertib. PDOs were sequenced (DNA & RNA) at multiple timepoints of treatment (3 days, 4 and 9 weeks after development of resistance). Using CRISPR-Cas9 editing we also engineered CAMA1 (ER+) cells to express AKT1 E17K, and generated derived resistant AKT1 mutant and parental models in response to continuous capivasertib exposure. CAMA1 AKT1 mutant and parental resistant were screened with an siRNA Kinome library supplemented with genes from the PDO RNA-sequencing. Phosphoproteomics was performed on capivasertib resistant E17K models in the presence and absence of capivasertib. PDO models persisting after either 4 or 9 weeks of capivasertib treatment showed no acquisition of genetic driver events, or changes in AKT copy number. Resistant organoids at 9 weeks treatment showed increased expression of ERBB3 (HER3), EGFR3 and LPAR5 as well as increased expression of MAPK signalling components. In siRNA screens on CAMA1 derived resistant models, siRNA targeting ERBB2, CDK4, PLK4 and CHEK1, ESR1 and LPAR5 modulated sensitivity to capivasertib. Analysis of phosphoproteins in capivasertib resistant models revealed reactivation of mTORC1 signalling despite sustained AKT inhibition, with dramatic upregulation of PDK1 and mTORC2. Consistent with the siRNA screens, drug combinations identified fulvestrant as a synergistic agent with capivasertib in the resistant E17K models. Sensitivity to mTOR inhibition was maintained in capivasertib resistant models, with mTOR inhibition re-sensitizing cells to capivasertib. In these models, acquired resistance to capivasertib occurs through upregulation of ER signalling and rewiring of pathway signalling to bypass AKT inhibition and reactivate mTORC1. Targeting these mechanisms could prolong the use of capivasertib, with future work focusing on validating these mechanisms and re-sensitizing drug combinations in other AKT1 mutant models & PDOs. Citation Format: Sarah Mearns, Alex Pearson, Li-Xuan Sim, Rosalind Cutts, Prithika Sritharan, Heena Shah, Aditi Gulati, Stuart Williamson, Simon Barry, Elza De-Bruin, Nicholas C. Turner. Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB450.

Abstract LB176: Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids

Abstract A major barrier in lung cancer pre-clinical research is the lack of simple, replicative and reproducible cell culture models. Most studies are still focused on 2D, completely ignoring cell-to-cell interactions, in vivo tissue architecture and the surrounding microenvironment. To address this void in cancer research, we aimed to develop a biomimetic 3D lung cancer spheroid model that can be used as a more reliable tool for pharmacological studies. Therefore, 3D spheroids of A549 (adenocarcinoma), H460 (large cell carcinoma) and H520 (squamous cell carcinoma) non-small cell lung cancer (NSCLC) cells were optimized and consequently submitted to AZD2014 treatment, a dual mTOR inhibitor targeting mTORC1 and mTORC2, members of the PI3K/Akt/mTOR pathway, which is commonly deregulated in lung cancer. Each cell line was seeded at 2000, 5000 or 10000 cells/spheroid in agarose micro-molds for eight days. The 3D NSCLC spheroids were further characterized for their tumor features, namely 3D dynamics and morphology, including diameter, circularity and compactness, viability properties, as well as proliferative and necrotic areas. Based on this characterization, the 2000 cells/spheroid condition was selected for further treatments and spheroid treatment time-points were selected. For each cell line, spheroids were treated with AZD2014, using concentrations based on the IC30 values (sensitization concentration) defined under 2D conditions: 10 μM, 10 μM and 0.2 μM for A549, H460 and H520 spheroids, respectively. Treated and untreated spheroids were followed during six days post-treatment, and their morphological characteristics, cell viability, necrotic core formation and proliferative areas, as well as ATP production, were assessed. Spheroids of all cell lines exhibited a significant inhibition of spheroid growth with AZD2014 treatment, which was maintained over the six days. Moreover, a reduction in spheroid intracellular ATP was observed at days three and six post-treatment. An additional treatment regimen was studied by re-treating spheroids with the same concentration of AZD2014 after 48hrs from the first treatment. Spheroids exposed to this dual treatment regimen exhibited a significant inhibition of spheroid growth, while the respective controls exhibited intensified growth. While AZD2014 did not induce significant reductions in 3D cell viability, a clear inhibition of spheroid growth was observed. Having a more intricate cellular organization, this study alleviates the increased complexity of pharmacological responses in 3D spheroids when compared to 2D models. Further studies are being carried out to define the enhanced biomimetic ability of these NSCLC spheroids and the underlying mechanisms involved when targeting the PI3K/Akt/mTOR pathway using AZD2014 as mono-therapy or in combination with two novel anti-sense oligonucleotides (ASO). Citation Format: Nathan Vella, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, Vanessa Petroni Magri. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB176.

Rapamycin Controls Lymphoproliferation and Reverses T-Cell Responses in a Patient with a Novel STIM1 Loss-of-Function Deletion

PurposeDeficiency of stromal interaction molecule 1 (STIM1) results in combined immunodeficiency accompanied by extra-immunological findings like enamel defects and myopathy. We here studied a patient with a STIM1 loss-of-function mutation who presented with severe lymphoproliferation. We sought to explore the efficacy of the mTOR inhibitor rapamycin in controlling disease manifestations and reversing aberrant T-cell subsets and functions, which has never been used previously in this disorder.MethodsClinical findings of the patient were collected over time. We performed immunological evaluations before and after initiation of rapamycin treatment, including detailed lymphocyte subset analyses, alterations in frequencies of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes as well as T cell activation and proliferation capacities.ResultsA novel homozygous exon 2 deletion in STIM1 was detected in a 3-year-old girl with severe lymphoproliferation, recurrent infections, myopathy, iris hypoplasia, and enamel hypoplasia. Lymphoproliferation was associated with severe T-cell infiltrates. The deletion resulted in a complete loss of protein expression, associated with a lack of store-operated calcium entry response, defective T-cell activation, proliferation, and cytokine production. Interestingly, patient blood contained fewer cTFH and increased circulating follicular regulatory (cTFR) cells. Abnormal skewing towards TH2-like responses in certain T-cell subpopulations like cTFH, non-cTFH memory T-helper, and Treg cells was associated with increased eosinophil numbers and serum IgE levels. Treatment with rapamycin controlled lymphoproliferation, improved T-cell activation and proliferation capacities, reversed T-cell responses, and repressed high IgE levels and eosinophilia.ConclusionsThis study enhances our understanding of STIM1 deficiency by uncovering additional abnormal T-cell responses, and reveals for the first time the potential therapeutic utility of rapamycin for this disorder.

Probiotic-derived silver nanoparticles target mTOR/MMP-9/BCL-2/dependent AMPK activation for hepatic cancer treatment

Recent advances in nanotechnology have offered novel ways to combat cancer. By utilizing the reducing capabilities of Lactobacillus acidophilus, silver nanoparticles (AgNPs) are synthesized. The anti-cancer properties of AgNPs have been demonstrated in previous studies against several cancer cell lines; it has been hypothesized that these compounds might inhibit AMPK/mTOR signalling and BCL-2 expression. Consequently, the current research used both in vitro and in silico approaches to study whether Lactobacillus acidophilus AgNPs could inhibit cell proliferation autophagy and promote apoptosis in HepG2 cells. The isolated strain was identified as Lactobacillus acidophilus strain RBIM based on 16 s rRNA gene analysis. Based on our research findings, it has been observed that this particular strain can generate increased quantities of AgNPs when subjected to optimal growing conditions. The presence of silanols, carboxylates, phosphonates, and siloxanes on the surface of AgNPs was confirmed using FTIR analysis. AgNPs were configured using UV–visible spectroscopy at 425 nm. In contrast, it was observed that apoptotic cells exhibited orange-coloured bodies due to cellular shrinkage and blebbing initiated by AgNP treatment, compared to non-apoptotic cells. It is worth mentioning that AgNPs exhibited remarkable selectivity in inducing cell death, specifically in HepG2 cells, unlike normal WI-38 cells. The half-maximum inhibitory concentration (IC50) values for HepG2 and WI-38 cells were 4.217 µg/ml and 154.1 µg/ml, respectively. AgNPs induce an upregulation in the synthesis of inflammation-associated cytokines, including (TNF-α and IL-33), within HepG2 cells. AgNPs co-treatment led to higher glutathione levels and activating pro-autophagic genes such as AMPK.Additionally, it resulted in the suppression of mTOR, MMP-9, BCL-2, and α-SMA gene expression. The docking experiments suggest that the binding of AgNPs to the active site of the AMPK enzyme leads to inhibiting its activity. The inhibition of AMPK ultimately results in the suppression of the mechanistic mTOR and triggers apoptosis in HepG2 cells. In conclusion, the results of our study indicate that the utilization of AgNPs may represent a viable strategy for the eradication of liver cancerous cells through the activation of apoptosis and the enhancement of immune system reactions.