Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models.

The EGFR tyrosine kinase inhibitor osimertinib has been approved for the first-line treatment of EGFR-mutated Non-Small Cell Lung Cancer (NSCLC) patients. Despite its efficacy, patients develop resistance. Mechanisms of resistance are heterogeneous and not fully understood, and their characterization is essential to find new strategies to overcome resistance. Ceramides are well-known regulators of apoptosis and are converted into glucosylceramides (GlcCer) by glucosylceramide synthase (GCS). A higher content of GlcCers was observed in lung pleural effusions from NSCLC patients and their role in osimertinib-resistance has not been documented. The aim of this study was to determine the therapeutic potential of inhibiting GCS in NSCLC EGFR-mutant models resistant to osimertinib in vitro and in vivo. Lipidomic analysis showed a significant increase in the intracellular levels of glycosylceramides, including GlcCers in osimertinib resistant clones compared to sensitive cells. In resistant cells, the GCS inhibitor PDMP caused cell cycle arrest, inhibition of 2D and 3D cell proliferation, colony formation and migration capability, and apoptosis induction. The intratumoral injection of PDMP completely suppressed the growth of OR xenograft models. This study demonstrated that dysregulation of ceramide metabolism is involved in osimertinib-resistance and targeting GCS may be a promising therapeutic strategy for patients progressed to osimertinib.

Targeting metabolic adaptive responses induced by glucose starvation inhibits cell proliferation and enhances cell death in osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines.

Osimertinib, a tyrosine kinase inhibitor targeting mutant EGFR, has received approval for initial treatment in patients with Non-Small Cell Lung Cancer (NSCLC). While effective in both first- and second-line treatments, patients eventually develop acquired resistance. Metabolic reprogramming represents a strategy through which cancer cells may resist and adapt to the selective pressure exerted by the drug. In the current study, we investigated the metabolic adaptations associated with osimertinib-resistance in NSCLC cells under low glucose culture conditions. We demonstrated that, unlike osimertinib-sensitive cells, osimertinib-resistant cells were able to survive under low glucose conditions by increasing the rate of glucose and glutamine uptake and by shifting towards mitochondrial metabolism. Inhibiting glucose/pyruvate contribution to mitochondrial respiration, glutamine deamination to glutamate, and oxidative phosphorylation decreased the proliferation and survival abilities of osimertinib-resistant cells to glucose starvation. Our findings underscore the remarkable adaptability of osimertinib-resistant NSCLC cells in a low glucose environment and highlight the pivotal role of mitochondrial metabolism in mediating this adaptation. Targeting the metabolic adaptive responses triggered by glucose shortage emerges as a promising strategy, effectively inhibiting cell proliferation and promoting cell death in osimertinib-resistant cells.

Intrinsic Resistance to Osimertinib in EGFR Mutated NSCLC Cell Lines Induced by Alteration in Cell-Cycle Regulators.

BACKGROUND: Cell-cycle regulators are mutated in approximately 40% of all cancer types and have already been linked to worse outcomes in non-small cell lung cancer adenocarcinomas treated with osimertinib. However, their exact role in osimertinib resistance has not been elucidated. OBJECTIVE: In this study, we aimed to evaluate how the CDK4/6-Rb axis may affect the sensitivity to osimertinib. METHODS: We genetically increased the level of CCND1 (Cyclin D1) and reduced the levels of CDKN2A (p16) in two different adenocarcinoma cell lines, PC9 and HCC827. We also retrospectively evaluated the outcome of patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer depending on their level of Cyclin D1 and p16. RESULTS: The modified clones showed higher proliferative capacity, modifications in cell-cycle phases, and higher migratory capacity than the parental cells. Cyclin D1-overexpressing clones were highly resistant to acute osimertinib treatment. CDKN2A knockdown conferred intrinsic resistance as well, although a longer time was required for adaption to the drug. In both cases, the resistant phenotype was epidermal growth factor receptor independent and associated with a higher level of Rb phosphorylation, which was unaffected by osimertinib treatment. Blocking the phosphorylation of Rb using abemaciclib, a CDK4/6 inhibitor, exerted an additive effect with osimertinib, increasing sensitivity to this drug and reverting the intrinsic resistant phenotype. In a group of 32 patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer, assessed for Cyclin D1 and p16 expression, we found that the p16-deleted group presented a lower overall response rate compared with the control group. CONCLUSIONS: We conclude that perturbation in cell-cycle regulators leads to intrinsic osimertinib resistance and worse patient outcomes.

Mesenchymal stromal cells loaded with Paclitaxel (PacliMES) a potential new therapeutic approach on mesothelioma.

Malignant pleural mesothelioma is an asbestos-related tumor originating in mesothelial cells of the pleura that poorly responds to chemotherapeutic approaches. Adult mesenchymal stromal cells derived either from bone marrow or from adipose tissue may be considered a good model for cell-based therapy, a treatment which has experienced significant interest in recent years. The present study confirms that Paclitaxel is effective on mesothelioma cell proliferation in 2D and 3D in vitro cultures, and that 80,000 mesenchymal stromal cells loaded with Paclitaxel inhibit tumor growth at a higher extent than Paclitaxel alone. An in vivo approach to treat in situ mesothelioma xenografts using a minimal amount of 106 mesenchymal stromal cells loaded with Paclitaxel showed the same efficacy of a systemic administration of 10 mg/kg of Paclitaxel. These data strongly support drug delivery system by mesenchymal stromal cells as a useful approach against many solid tumors. We look with interest at the favourable opinion recently expressed by the Italian Drug Agency on the procedure for the preparation of mesenchymal stromal cells loaded with Paclitaxel in large-scale bioreactor systems and their storage until clinical use. This new Advanced Medicinal Therapy Product, already approved for a Phase I clinical trial on mesothelioma patients, could pave the way for mesenchymal stromal cells use as drug delivery system on other solid tumors for adjuvant therapy associated with surgery and radiotherapy.

Interaction between Occupational and Non-Occupational Arsenic Exposure and Tobacco Smoke on Lung Cancerogenesis: A Systematic Review.

Although a higher lung cancer risk has been already associated with arsenic exposure, the contribution of arsenic and its compounds to the carcinogenic effects of other agents, such as tobacco smoke, is not well characterized. This systematic review examined the relationship between occupational and non-occupational arsenic exposure and tobacco smoking on lung cancer risk using papers published from 2010 to 2022. Two databases, PUBMED and Scifinder, were used for the searches. Among the sixteen human studies included, four were about occupational exposure, and the others were about arsenic in drinking water. Furthermore, only three case-control studies and two cohort studies evaluated an additive or multiplicative interaction. The interaction between arsenic exposure and tobacco smoke seems to be negligible at low arsenic concentrations (<100 µg/L), while there is a synergistic effect at higher concentrations. Finally, it is not yet possible to assess whether a linear no-threshold (LNT) model for lung cancer risk can be applied to the co-exposure to arsenic and tobacco smoke. Although the methodological quality of the included studies is good, these findings suggest that rigorous and accurate prospective studies on this topic are highly needed.

Micro-fragmented Fat Inhibits the Progression of Human Mesothelioma Xenografts in Mice.

BACKGROUND: Malignant pleural mesothelioma is a pathology with no effective therapy and a poor prognosis. Our previous study demonstrated an in vitro inhibitory effect on mesothelioma cell lines of both the lysate and secretome of adipose tissue-derived Mesenchymal Stromal Cells. The inhibitory activity on tumor growth has been demonstrated also in vivo: five million Mesenchymal Stromal Cells, injected "in situ", produced a significant therapeutic efficacy against MSTO-211H xenograft equivalent to that observed after the systemic administration of paclitaxel. OBJECTIVE: The objective of this study is to evaluate the efficacy of low amount (half a million) Mesenchymal Stromal Cells and micro-fragmented adipose tissues (the biological tissue from which the Mesenchymal Stromal Cells were isolated) on mesothelioma cells growth. METHODS: Tumor cells growth inhibition was evaluated in vitro and in a xenograft model of mesothelioma. RESULTS: The inhibitory effect of micro-fragmented fat from adipose-tissue has been firstly confirmed in vitro on MSTO-211H cell growth. Then the efficacy against the growth of mesothelioma xenografts in mice of both micro-fragmented fat and low amount of Mesenchymal Stromal Cells has been evaluated. Our results confirmed that both Mesenchymal Stromal Cells and micro-fragmented fat, injected "in situ", did not stimulate mesothelioma cell growth. By contrast, micro-fragmented fat produced a significant inhibition of tumor growth and progression, comparable to that observed by the treatment with paclitaxel. Low amount of Mesenchymal Stromal Cells exerted only a little anticancer activity. CONCLUSION: Micro-fragmented fat inhibited mesothelioma cell proliferation in vitro and exerted a significant control of the mesothelioma xenograft growth in vivo.

A New ABCB1 Inhibitor Enhances the Anticancer Effect of Doxorubicin in Both In Vitro and In Vivo Models of NSCLC.

In tumors, the multi drug resistance phenomenon may occur through the efflux of chemotherapeutic drugs out of cancer cells, impeding their accumulation, and eventually reducing their toxicity. This process is mediated by transporters overexpressed in the plasma membranes of tumor cells, among which is the P-glycoprotein/multidrug resistance 1/ATP-binding cassette B1 (P-gp/MDR1/ABCB1). The aim of this study was to explore the effect of a new molecule, called AIF-1, on ABCB1 activity. In a cellular model of non-small cell lung cancer (NSCLC), AIF-1 significantly inhibited ABCB1 activity, which was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein. AIF-1 also significantly increased the intracellular content of doxorubicin, which was evaluated by confocal microscopy and LC-MS/MS analysis. This effect translated to higher cytotoxicity of doxorubicin and reduced cellular proliferation. Finally, in a murine xenograft model, the tumor volume increased by 267% and 148% on average in mice treated with vehicle and doxorubicin alone, respectively. After the co-administration of doxorubicin with AIF-1, tumor volume increased by only 13.4%. In conclusion, these results suggest enhancement of the efficacy of the chemotherapeutic drug doxorubicin by AIF-1, laying the basis for the future development of new ABCB1 inhibitors for tumor treatment.

CDK4/6 Inhibition Enhances the Efficacy of Standard Chemotherapy Treatment in Malignant Pleural Mesothelioma Cells.

BACKGROUND: The loss of the CDKN2A/ARF (cyclin-dependent kinase inhibitor 2A/alternative reading frame) gene is the most common alteration in malignant pleural mesothelioma (MPM), with an incidence of about 70%, thus representing a novel target for mesothelioma treatment. In the present study, we evaluated the antitumor potential of combining the standard chemotherapy regimen used for unresectable MPM with the CDK4/6 (cyclin-dependent kinase 4 or 6) inhibitor abemaciclib. METHODS: Cell viability, cell death, senescence, and autophagy induction were evaluated in two MPM cell lines and in a primary MPM cell culture. RESULTS: The simultaneous treatment of abemaciclib with cisplatin and pemetrexed showed a greater antiproliferative effect than chemotherapy alone, both in MPM cell lines and in primary cells. This combined treatment induced cellular senescence or autophagic cell death, depending on the cell type. More in detail, the induction of cellular senescence was related to the increased expression of p21, whereas autophagy induction was due to the impairment of the AKT/mTOR signaling. Notably, the effect of the combination was irreversible and no resumption in tumor cell proliferation was observed after drug withdrawal. CONCLUSION: Our results demonstrated the therapeutic potential of CDK4/6 inhibitors in combination with chemotherapy for the treatment of MPM and are consistent with the recent positive results in the MiST2 arm in abemaciclib-treated patients.

CDK4/6 inhibitors improve the anti-tumor efficacy of lenvatinib in hepatocarcinoma cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer with a poor prognosis and limited treatment options. Considering that alterations of the CDK4/6-cyclin D-Rb pathway occur frequently in HCC, we tested the efficacy of two CDK4/6 inhibitors, abemaciclib and ribociclib, in combination with lenvatinib, a multi-kinase inhibitor approved as first-line therapy for advanced HCC, in a panel of HCC Rb-expressing cell lines. The simultaneous drug combinations showed a superior anti-proliferative activity as compared with single agents or sequential schedules of treatment, either in short or in long-term experiments. In addition, the simultaneous combination of abemaciclib with lenvatinib reduced 3D cell growth, and impaired colony formation and cell migration. Mechanistically, these growth-inhibitory effects were associated with a stronger down-regulation of c-myc protein expression. Depending on the HCC cell model, reduced activation of MAPK, mTORC1/p70S6K or src/FAK signaling was also observed. Abemaciclib combined with lenvatinib arrested the cells in the G1 cell cycle phase, induced p21 accumulation, and promoted a stronger increase of cellular senescence, associated with elevation of ß-galactosidase activity and accumulation of ROS, as compared with single treatments. After drug withdrawal, the capacity of forming colonies was significantly impaired, suggesting that the anti-tumor efficacy of abemaciclib and lenvatinib combination was persistent. Our pre-clinical results demonstrate the effectiveness of the simultaneous combination of CDK4/6 inhibitors with lenvatinib in HCC cell models, suggesting that this combination may be worthy of further investigation as a therapeutic approach for the treatment of advanced HCC.

Interaction between occupational radon exposure and tobacco smoke: a systematic review.

INTRODUCTION: The risk of lung cancer from radon exposure was small compared to tobacco smoking (BEIR VI), but the relationship between these two carcinogenic agents has yet to be quantitatively estimated. The objective of this systematic review was to evaluate the last evidences on the role of radon occupational exposures and tobacco smoke on lung cancer risk. AREAS COVERED: Thirteen articles were selected using two different databases, PubMed and Scifinder, and were limited to those published from 2010 to 2021. The reference list of selected studies was reviewed to identify other relevant papers. EXPERT OPINION: Seven papers included in this systematic review did not deal with the multiplicative or the additive type of interaction between radon exposure and smoking habit. Six papers discussed the nature of this interaction with a prevalence of the sub-multiplicative model compared to the additive one. Altogether, smoking adjustment did not significantly change lung cancer risk. The included studies might constitute a starting point for updating the models for risk assessment in occupational and residential scenarios, promoting concomitantly the exposure reduction to radon and other cofactors, as recently introduced by Italian Legislative Decree number 101 of 31 July 2020, an application of Euratom Directive 59/2013.

This paper offers an updated overview on lung cancer risk due to occupational exposure to radon together with tobacco smoking habits. It evidences the quantitative role of smoking on radon exposure and discusses their interaction. The results show that smoking adjustments do not significantly modify lung cancer risk at both high and low radon concentrations.

EGFR Signaling in Non-Small Cell Lung Cancer: From Molecular Mechanisms to Therapeutic Opportunities.

Lung cancer is the leading cause of cancer death worldwide [...].

Reprogramming of Lipid Metabolism in Lung Cancer: An Overview with Focus on EGFR-Mutated Non-Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Most of lung cancer cases are classified as non-small cell lung cancers (NSCLC). EGFR has become an important therapeutic target for the treatment of NSCLC patients, and inhibitors targeting the kinase domain of EGFR are currently used in clinical settings. Recently, an increasing interest has emerged toward understanding the mechanisms and biological consequences associated with lipid reprogramming in cancer. Increased uptake, synthesis, oxidation, or storage of lipids has been demonstrated to contribute to the growth of many types of cancer, including lung cancer. In this review, we provide an overview of metabolism in cancer and then explore in more detail the role of lipid metabolic reprogramming in lung cancer development and progression and in resistance to therapies, emphasizing its connection with EGFR signaling. In addition, we summarize the potential therapeutic approaches targeting lipid metabolism for lung cancer treatment.

YES1 and MYC Amplifications as Synergistic Resistance Mechanisms to Different Generation ALK Tyrosine Kinase Inhibitors in Advanced NSCLC: Brief Report of Clinical and Preclinical Proofs.

INTRODUCTION: ALK tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced ALK-positive NSCLC. Nevertheless, drug resistance inevitably occurs. Here, we report a case of a patient with metastatic ALK-positive lung adenocarcinoma with an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplification in a contest of epithelial-to-mesenchymal transition and high progressive chromosomal instability. METHODS: The patient received, after chemotherapy and 7 months of crizotinib, brigatinib and lorlatinib with no clinical benefit to both treatments. A study of resistance mechanisms was performed with whole exome sequencing on different biological samples; primary cell lines were established from pleural effusion after lorlatinib progression. RESULTS: At whole exome sequencing analysis, YES1 and MYC amplifications were observed both in the pericardial biopsy and the pleural effusion samples collected at brigatinib and lorlatinib progression, respectively. Increasing chromosomal instability from diagnostic biopsy to pleural effusion was also observed. The addition of dasatinib to brigatinib or lorlatinib restored the sensitivity in primary cell lines; data were confirmed also in H3122_ALK-positive model overexpressing both YES1 and MYC. CONCLUSIONS: In conclusion, YES1 and MYC amplifications are candidates to justify a rapid acquired resistance to crizotinib entailing primary brigatinib and lorlatinib resistance. In this context, a combination strategy of ALK TKI with dasatinib could be effective to overcome a rapid resistance.

A sulfonyl fluoride derivative inhibits EGFRL858R/T790M/C797S by covalent modification of the catalytic lysine.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.

Generation and Characterization of a New Preclinical Mouse Model of EGFR-Driven Lung Cancer with MET-Induced Osimertinib Resistance.

Despite the introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to treat advanced lung cancer harboring EGFR-activating mutations, the prognosis remains unfavorable because of intrinsic and/or acquired resistance. We generated a new state-of-the-art mouse strain harboring the human EGFRT790M/L858R oncogene and MET overexpression (EGFR/MET strain) that mimics the MET amplification occurring in one out of five patients with EGFR-mutated lung cancer that relapsed after treatment with osimertinib, a third-generation anti-EGFR TKI. We found that survival was reduced in EGFR/MET mice compared with mice harboring only EGFRT790M/L858R (EGFR strain). Moreover, EGFR/MET-driven lung tumors were resistant to osimertinib, recapitulating the phenotype observed in patients. Conversely, as also observed in patients, the crizotinib (anti-MET TKI) and osimertinib combination improved survival and reduced tumor burden in EGFR/MET mice, further validating the model's value for preclinical studies. We also found that in EGFR/MET mice, MET overexpression negatively regulated EGFR activity through MIG6 induction, a compensatory mechanism that allows the coexistence of the two onco-genic events. Our data suggest that single EGFR or MET inhibition might not be a good therapeutic option for EGFR-mutated lung cancer with MET amplification, and that inhibition of both pathways should be the best clinical choice in these patients.

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

Fighting tertiary mutations in EGFR-driven lung-cancers: Current advances and future perspectives in medicinal chemistry.

Third-generation inhibitors of the epidermal growth factor receptor (EGFR), best exemplified by osimertinib, have been developed to selectively target variants of EGFR bearing activating mutations and the mutation of gatekeeper T790 in patients with EGFR-mutated forms of Non-Small Cell Lung Cancer (NSCLC). While the application of third-generation inhibitors has represented an effective first- and second-line treatment, the efficacy of this class of inhibitors has been hampered by the novel, tertiary mutation C797S, which may occur after the treatment with osimertinib. More recently, other point mutations, including L718Q, G796D, G724S, L792 and G719, have emerged as mutations mediating resistance to third-generation inhibitors. The challenge of overcoming newly developed and recurrent resistances mediated by EGFR-mutations is thus driving the search of alternative strategies in the design of new therapeutic agents able to block EGFR-driven tumor growth. In this manuscript we review the recently emerged EGFR-dependent mechanisms of resistance to third-generation inhibitors, and the achievements lately obtained in the development of next-generation EGFR inhibitors.

Small Cell Lung Cancer Transformation as a Resistance Mechanism to Osimertinib in Epidermal Growth Factor Receptor-Mutated Lung Adenocarcinoma: Case Report and Literature Review.

Introduction: Small cell lung cancer (SCLC) transformation represents a mechanism of resistance to osimertinib in EGFR-mutated lung adenocarcinoma, which dramatically impacts patients' prognosis due to high refractoriness to conventional treatments. Case Description: We present the case of a patient who developed a SCLC phenotypic transformation as resistance mechanism to second-line osimertinib for T790M-positive EGFR-mutated NSCLC. Our patient received platinum-etoposide doublet following SCLC switch and achieved a modest clinical benefit which lasted 4 months. NGS and IHC analyses for p53 and Rb were performed on subsequent liver biopsies, revealing baseline TP53 mutation and complete absence of p53 and Rb expression. Primary cell cultures were established following a liver biopsy at the time of SCLC transformation, and drug sensitivity assays showed meaningful cell growth inhibition when osimertinib was added to platinum-etoposide compared with control (p < 0.05). A review of the current literature regarding SCLC transformation after failure of osimertinib was performed. Conclusions: Based on retrospective data available to date, platinum-etoposide chemotherapy is the preferred treatment choice in the occurrence of SCLC transformation after osimertinib failure. The extension of osimertinib in combination with chemotherapy in the occurrence of SCLC transformation as resistance mechanism to osimertinib is a matter of debate. The combination of osimertinib and platinum-etoposide was effective in inhibiting cell growth in our primary cell cultures. Clinical studies are needed to further explore this combination in the occurrence of SCLC transformation as a resistance mechanism to osimertinib.

Afatinib therapy in case of EGFR G724S emergence as resistance mechanism to osimertinib.

Osimertinib is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) used both as the first-line treatment of EGFR-mutated non-small cell lung cancer patients and in second-line after T790M-positive disease progression to first- or second-generation TKIs. Unfortunately, patients unavoidably experience disease progression to osimertinib and the current research is focused on resistance mechanisms and the relative therapeutic strategy. We report the case of a patient with advanced EGFR-mutated (exon 19 deletion and T790M-positive) non-small cell lung cancer who developed disease progression to osimertinib characterized by the loss of T790M concurrently with the emergence of G724S EGFR mutation, which was tackled by subsequent afatinib treatment. Next-generation sequencing molecular study of rebiopsy at time of progression to osimertinib revealed the persistence of EGFR exon 19 deletion, loss of T790M with a new G724S EGFR mutation; other concomitant mechanisms were excluded. Retrospective analysis of cell-free DNA revealed the emergence of G724S EGFR mutation four months before the radiologically-proven disease progression. The patient, after chemotherapy, was treated with afatinib with clinical and radiological benefit. Our case report contributes to increase the knowledge on acquired resistance mechanisms to osimertinib treatment, and it shows, for the first time, the efficacy of afatinib in the case of T790M loss and emergence of G724S EGFR mutation.

Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib.

Abemaciclib is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 that inhibits the transition from the G1 to the S phase of the cell cycle by blocking downstream CDK4/6-mediated phosphorylation of Rb. The effects of abemaciclib alone or combined with the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib were examined in a panel of PC9 and HCC827 osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines carrying EGFR-dependent or -independent mechanisms of intrinsic or acquired resistance. Differently from sensitive cells, all the resistant cell lines analyzed maintained p-Rb, which may be considered as a biomarker of osimertinib resistance and a potential target for therapeutic intervention. In these models, abemaciclib inhibited cell growth, spheroid formation, colony formation, and induced senescence, and its efficacy was not enhanced in the presence of osimertinib. Interestingly, in osimertinib sensitive PC9, PC9T790M, and H1975 cells the combination of abemaciclib with osimertinib significantly inhibited the onset of resistance in long-term experiments. Our findings provide a preclinical support for using abemaciclib to treat resistance in EGFR mutated NSCLC patients progressed to osimertinib either as single treatment or combined with osimertinib, and suggest the combination of osimertinib with abemaciclib as a potential approach to prevent or delay osimertinib resistance in first-line treatment.