PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Selective Termination of Autophagy-Dependent Cancers.

The goal of cancer research is to identify characteristics of cancer cells that allow them to be selectively eliminated without harming the host. One such characteristic is autophagy dependence. Cancer cells survive, proliferate, and metastasize under conditions where normal cells do not. Thus, the requirement in cancer cells for more energy and macromolecular biosynthesis can evolve into a dependence on autophagy for recycling cellular components. Recent studies have revealed that autophagy, as well as different forms of cellular trafficking, is regulated by five phosphoinositides associated with eukaryotic cellular membranes and that the enzymes that synthesize them are prime targets for cancer therapy. For example, PIKFYVE inhibitors rapidly disrupt lysosome homeostasis and suppress proliferation in all cells. However, these inhibitors selectively terminate PIKFYVE-dependent cancer cells and cancer stem cells with not having adverse effect on normal cells. Here, we describe the biochemical distinctions between PIKFYVE-sensitive and -insensitive cells, categorize PIKFYVE inhibitors into four groups that differ in chemical structure, target specificity and efficacy on cancer cells and normal cells, identify the mechanisms by which they selectively terminate autophagy-dependent cancer cells, note their paradoxical effects in cancer immunotherapy, and describe their therapeutic applications against cancers.

Designing Small Molecule PI3Kγ Inhibitors: A Review of Structure-Based Methods and Computational Approaches.

The PI3K/AKT/mTOR pathway plays critical roles in a wide array of biological processes. Phosphatidylinositol 3-kinase gamma (PI3Kγ), a class IB PI3K family member, represents a potential therapeutic opportunity for the treatment of cancer, inflammation, and autoimmunity. In this Perspective, we provide a comprehensive overview of the structure, biological function, and regulation of PI3Kγ. We also focus on the development of PI3Kγ inhibitors over the past decade and emphasize their binding modes, structure-activity relationships, and pharmacological activities. The application of computational technologies and artificial intelligence in the discovery of novel PI3Kγ inhibitors is also introduced. This review aims to provide a timely and updated overview on the strategies for targeting PI3Kγ.

Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer.

Colorectal cancer (CRC) is one of the most common tumors of the digestive system worldwide. KRAS mutations limit the use of anti-EGFR antibodies in combination with chemotherapy for the treatment of CRC. Therefore, novel targeted therapies are needed to overcome the KRAS-induced oncogenesis. Recent evidence suggests that inhibition of PI3K led to ferroptosis, a nonapoptotic cell death closely related to KRAS-mutant cells. Here, we showed that a selective PI3Kδ inhibitor TYM-3-98 can suppress the AKT/mTOR signaling and activate the ferroptosis pathway in KRAS-mutant CRC cells in a concentration-dependent manner. This was evidenced by the lipid peroxidation, iron accumulation, and depletion of GSH. Moreover, the overexpression of the sterol regulatory element-binding protein 1 (SREBP1), a downstream transcription factor regulating lipid metabolism, conferred CRC cells greater resistance to ferroptosis induced by TYM-3-98. In addition, the effect of TYM-3-98 was confirmed in a xenograft mouse model, which demonstrated significant tumor suppression without obvious hepatoxicity or renal toxicity. Taken together, our work demonstrated that the induction of ferroptosis contributed to the PI3Kδ inhibitor-induced cell death via the suppression of AKT/mTOR/SREBP1-mediated lipogenesis, thus displaying a promising therapeutic effect of TYM-3-98 in CRC treatment.

Therapeutic potential of inhibiting the PI3Kγ complex for leukemia.

In their recent paper published in Nature, Luo et al.1 investigate the cancer-cell-intrinsic roles of the PI3Kγ complex in leukemia. Their findings pinpoint PI3Kγ inhibition as a possible novel treatment avenue for a subset of acute leukemias.

Combination Therapy Approach to Overcome the Resistance to PI3K Pathway Inhibitors in Gynecological Cancers.

The PI3K signaling pathway plays an essential role in cancer cell proliferation and survival. PI3K pathway inhibitors are now FDA-approved as a single agent treatment or in combination for solid tumors such as renal cell carcinoma or breast cancer. However, despite the high prevalence of PI3K pathway alterations in gynecological cancers and promising preclinical activity in endometrial and ovarian cancer models, PI3K pathway inhibitors showed limited clinical activity in gynecological cancers. In this review, we provide an overview on resistance mechanisms against PI3K pathway inhibitors that limit their use in gynecological malignancies, including genetic alterations that reactivate the PI3K pathway such as PIK3CA mutations and PTEN loss, compensatory signaling pathway activation, and feedback loops causing the reactivation of the PI3K signaling pathway. We also discuss the successes and limitations of recent clinical trials aiming to address such resistance mechanisms through combination therapies.

Paediatric strategy forum for medicinal product development of PI3-K, mTOR, AKT and GSK3ß inhibitors in children and adolescents with cancer.

Phosphatidylinositol 3-kinase (PI3-K) signalling pathway is a crucial path in cancer for cell survival and thus represents an intriguing target for new paediatric anti-cancer drugs. However, the unique clinical toxicities of targeting this pathway (resulting in hyperglycaemia) difficulties combining with chemotherapy, rarity of mutations in childhood tumours and concomitant mutations have resulted in major barriers to clinical translation of these inhibitors in treating both adults and children. Mutations in PIK3CA predict response to PI3-K inhibitors in adult cancers. The same mutations occur in children as in adults, but they are significantly less frequent in paediatrics. In children, high-grade gliomas, especially diffuse midline gliomas (DMG), have the highest incidence of PIK3CA mutations. New mutation-specific PI3-K inhibitors reduce toxicity from on-target PI3-Kα wild-type activity. The mTOR inhibitor everolimus is approved for subependymal giant cell astrocytomas. In paediatric cancers, mTOR inhibitors have been predominantly evaluated by academia, without an overall strategy, in empiric, mutation-agnostic clinical trials with very low response rates to monotherapy. Therefore, future trials of single agent or combination strategies of mTOR inhibitors in childhood cancer should be supported by very strong biological rationale and preclinical data. Further preclinical evaluation of glycogen synthase kinase-3 beta inhibitors is required. Similarly, even where there is an AKT mutation (∼0.1 %), the role of AKT inhibitors in paediatric cancers remains unclear. Patient advocates strongly urged analysing and conserving data from every child participating in a clinical trial. A priority is to evaluate mutation-specific, central nervous system-penetrant PI3-K inhibitors in children with DMG in a rational biological combination. The choice of combination, should be based on the genomic landscape e.g. PTEN loss and resistance mechanisms supported by preclinical data. However, in view of the very rare populations involved, innovative regulatory approaches are needed to generate data for an indication.

Discovery and Optimization of Pyridazinones as PI3Kδ Selective Inhibitors for Administration by Inhalation.

A hit-to-lead campaign pursuing the identification of novel inhalant small-molecule phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of inflammatory respiratory diseases is disclosed. A synthetically versatile pyridazin-3(2H)-one scaffold was designed, and three exit vectors on the core moiety were used to explore chemical diversity and optimize pharmacological and absorption, distribution, metabolism, and excretion (ADME) properties. Desired modulation of PI3Kδ selectivity and cellular potency as well as ADME properties in view of administration by inhalation was achieved. Intratracheal administration of lead compound 26 resulted in a promising pharmacokinetic profile, thus demonstrating that the optimization strategy of in vitro profiles successfully translated to an in vivo setting.

A comprehensive review on phosphatidylinositol-3-kinase (PI3K) and its inhibitors bearing pyrazole or indazole core for cancer therapy.

Cancer is a complex and multifaceted group of diseases with a high mortality rate characterized by uncontrolled proliferation of abnormal cells. Dysregulation of normal signalling pathways in cancer contributes to the different hallmarks of this disease. The signalling pathway of which phosphatidylinositol 3-kinase (PI3K) is a part is not an exception. In fact, dysregulated activation of PI3K signalling pathways can result in unbridled cellular proliferation and enhanced cell survival, thereby fostering the onset and advancement of cancer. Therefore, there is substantial interest in developing targeted therapies specifically aimed at inhibiting the PI3K enzyme and its associated pathways. Also, the therapeutic interest on pyrazoles and indazoles has been growing due to their various medicinal properties, namely, anticancer activity. Derivatives of these compounds have been studied as PI3K inhibitors, and they showed promising results. There are already some PI3K inhibitors approved by Food and Drug Administration (FDA), such as Idelalisib (Zydelig®) and Alpelisib (Piqray®). In this context, this review aims to address the importance of PI3K in cellular processes and its role in cancer. Additionally, it aims to report a comprehensive literature review of PI3K inhibitors, containing the pyrazole and indazole scaffolds, published in the last fifteen years, focusing on structure-activity relationship aspects, thus providing important insights for the design of novel and more effective PI3K inhibitors.

Inhibition of PIKfyve Leads to Lysosomal Disorders via Dysregulation of mTOR Signaling.

PIKfyve is an endosomal lipid kinase that synthesizes phosphatidylinositol 3,5-biphosphate from phosphatidylinositol 3-phsphate. Inhibition of PIKfyve activity leads to lysosomal enlargement and cytoplasmic vacuolation, attributed to impaired lysosomal fission processes and homeostasis. However, the precise molecular mechanisms underlying these effects remain a topic of debate. In this study, we present findings from PIKfyve-deficient zebrafish embryos, revealing enlarged macrophages with giant vacuoles reminiscent of lysosomal storage disorders. Treatment with mTOR inhibitors or effective knockout of mTOR partially reverses these abnormalities and extend the lifespan of mutant larvae. Further in vivo and in vitro mechanistic investigations provide evidence that PIKfyve activity is essential for mTOR shutdown during early zebrafish development and in cells cultured under serum-deprived conditions. These findings underscore the critical role of PIKfyve activity in regulating mTOR signaling and suggest potential therapeutic applications of PIKfyve inhibitors for the treatment of lysosomal storage disorders.

PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

Novel isoniazid-hydrazone derivatives induce cell growth inhibition, cell cycle arrest and apoptosis via mitochondria-dependent caspase activation and PI3K/AKT inhibition.

In this study, seven isoniazid-hydrazone derivatives (3a-g) were synthesized and their structures elucidated by chromatographic techniques, and then the antiproliferative effects of these compounds on various cancer cells were tested. The advanced anticancer mechanism of the most potent compound was then investigated. Antiproliferative activities of the synthesized compounds were evaluated on human breast cancer MCF-7, lung cancer A-549, colon cancer HT-29, and non-cancerous mouse fibroblast 3T3-L1 cell lines by XTT assay. Flow cytometry analysis were carried out to determine cell cycle distribution, apoptosis, mitochondrial membrane potential, multi-caspase activity, and expression of PI3K/AKT signaling pathway. The XTT results showed that all the title molecules displayed cytotoxic activity at varying strengths in different dose ranges, and among them, the strongest cytotoxic effect and high selectivity were exerted by 3d against MCF-7 cells with the IC50 value of 11.35 µM and selectivity index of 8.65. Flow cytometry results revealed that compound 3d induced apoptosis through mitochondrial membrane disruption and multi-caspase activation in MCF-7 cells. It also inhibited the cell proliferation via inhibition of expression of PI3K/AKT and arrested the cell cycle at G0/G1 phase. In conclusion, all these data disclosed that among the synthesized compounds, 3d is notable for in vivo anticancer studies.

EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2)-negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2-negative, RAS wild-type mGAC patients.

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells.

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target.

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

The PI3K pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple-negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the antitumor effect in TNBC. A panel of eight TNBC patient-derived xenograft (PDX) models was tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment-induced signaling changes were examined by reverse phase protein array, immunohistochemistry (IHC) and 18F-fluorodeoxyglucose PET (18F-FDG PET). Compared with each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin-induced mitotic arrest. The combination enhanced induction of apoptosis compared with each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan and AKT phosphorylation by IHC. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin and led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC. SIGNIFICANCE: In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Navigating the complexity of PI3K/AKT pathway in HER-2 negative breast cancer: biomarkers and beyond.

The results of the SOLAR-1 and CAPItello-291, highlight the benefit of the ɑ-selective phosphoinositide 3-Kinase Pathway inhibitor (PI3Ki) alpelisib and the AKT inhibitor (AKTi) capivasertib in patients with hormone receptor-positive (HR+)/Human Epidermal Growth Factor Receptor 2 (HER2)- negative metastatic breast cancer (mBC) that have PIK3CA/AKT1/PTEN tumour alterations. Although effective, these drugs are associated with significant toxicities, which often limit their use, particularly in frail patients. Following the recent incorporation of these agents into clinical practice, and with many others currently in development, significant challenges have emerged, particularly those regarding biomarkers for patient selection. This review will discuss biomarkers of response and their resistance to PI3K/AKT inhibitors (PI3K/AKTis) in HR+/HER- BC in early and advanced settings to ascertain which populations will most benefit from these drugs. Of the biomarkers that were analysed, such as PIK3CA, AKT, PTEN mutations, insulin levels, 18 F-FDG-PET/TC, only the PIK3CA-mutations (PIK3CA-mut) and the AKT pathway alterations seem to have a predictive value for treatments with alpelisib and capivasertib. However, due to the retrospective and exploratory nature of the study, the data did not provide conclusive results. In addition, the different methods used to detect PIK3CA/AKT1/PTEN alterations underline the fact that the optimal diagnostic companion has yet to be established. We have summarised the clinical data on the approved and discontinued agents targeting this pathway and have assessed the drugs development, successes, and failures. Finally, because of tumour heterogeneity, we emphasise the importance of reassessing the mutational status of PI3KCA in both metastatic tissue and blood at the time of disease progression to better tailor treatment for patients.

PI3K inhibitor "alpelisib" alleviates methotrexate induced liver injury in mice and potentiates its cytotoxic effect against MDA-MB-231 triple negative breast cancer cell line.

Hepatotoxicity is the main off-target effect of methotrexate (MTX) limiting its effective clinical use. Besides, MDA-MB231 breast cancer cells show chemoresistance, partly via PI3K/AKT pathway. Therefore, we investigated the ameliorative potentials of the PI3K inhibitor, alpelisib (ALP) on MTX-induced hepatotoxicity (in vivo) and the restraining potentials of ALP on MDA-MB231 chemoresistance to MTX (in vitro). Twenty-eight male BALB/c mice were divided into 4 groups. In treatment groups, mice were administered ALP (2.5 and 5 mg/kg) for 5 days and MTX (20 mg/kg) from day 2 till day 5. The results showed that ALP restored hepatic architecture, reduced immune cell infiltration (F4/80, Ly6G and MPO) and repressed the rise in liver enzymes (AST and ALT) induced by MTX. Additionally, ALP rectified the MTX-induced disruption of cellular oxidant status by boosting antioxidant defense systems (HO-1 and GSH) and repressing lipid peroxidation (MDA and 4-HNE). Finally, ALP curbed MTX-induced hepatocyte apoptosis (NF-κB and BAX) and shifted the cytokine milieu away from inflammation (IL-17, IL-22, IL-6 and IL- 10). The results of the in vitro experiments revealed that ALP alone and in combination with MTX, synergistically, reduced cancer cell viability (MTT assay), migration (wound healing assay) and their capacity to establish colonies (colony formation assay) as compared to MTX alone. RT-PCR revealed the antiproliferative (Bcl-2) and proapoptotic (BAX) potentials of ALP and ALP/MTX combination especially after 24 h. In conclusion, targeting PI3K/AKT pathway is a promising strategy in triple negative breast cancer patients by ameliorating hepatotoxicity and restraining chemoresistance to chemotherapy.