NSCLC Cells Resistance to PI3K/mTOR Inhibitors Is Mediated by Delta-6 Fatty Acid Desaturase (FADS2).

Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most common events in human cancers. Several efforts have been made toward the identification of selective PI3K pathway inhibitors. However, the success of these molecules has been partially limited due to unexpected toxicities, the selection of potentially responsive patients, and intrinsic resistance to treatments. Metabolic alterations are intimately linked to drug resistance; altered metabolic pathways can help cancer cells adapt to continuous drug exposure and develop resistant phenotypes. Here we report the metabolic alterations underlying the non-small cell lung cancer (NSCLC) cell lines resistant to the usual PI3K-mTOR inhibitor BEZ235. In this study, we identified that an increased unsaturation degree of lipid species is associated with increased plasma membrane fluidity in cells with the resistant phenotype and that fatty acid desaturase FADS2 mediates the acquisition of chemoresistance. Therefore, new studies focused on reversing drug resistance based on membrane lipid modifications should consider the contribution of desaturase activity.

LKB1: Can We Target an Hidden Target? Focus on NSCLC.

LKB1 (liver kinase B1) is a master regulator of several processes such as metabolism, proliferation, cell polarity and immunity. About one third of non-small cell lung cancers (NSCLCs) present LKB1 alterations, which almost invariably lead to protein loss, resulting in the absence of a potential druggable target. In addition, LKB1-null tumors are very aggressive and resistant to chemotherapy, targeted therapies and immune checkpoint inhibitors (ICIs). In this review, we report and comment strategies that exploit peculiar co-vulnerabilities to effectively treat this subgroup of NSCLCs. LKB1 loss leads to an enhanced metabolic avidity, and treatments inducing metabolic stress were successful in inhibiting tumor growth in several preclinical models. Biguanides, by compromising mitochondria and reducing systemic glucose availability, and the glutaminase inhibitor telaglenastat (CB-839), inhibiting glutamate production and reducing carbon intermediates essential for TCA cycle progression, have provided the most interesting results and entered different clinical trials enrolling also LKB1-null NSCLC patients. Nutrient deprivation has been investigated as an alternative therapeutic intervention, giving rise to interesting results exploitable to design specific dietetic regimens able to counteract cancer progression. Other strategies aimed at targeting LKB1-null NSCLCs exploit its pivotal role in modulating cell proliferation and cell invasion. Several inhibitors of LKB1 downstream proteins, such as mTOR, MEK, ERK and SRK/FAK, resulted specifically active on LKB1-mutated preclinical models and, being molecules already in clinical experimentation, could be soon proposed as a specific therapy for these patients. In particular, the rational use in combination of these inhibitors represents a very promising strategy to prevent the activation of collateral pathways and possibly avoid the potential emergence of resistance to these drugs. LKB1-null phenotype has been correlated to ICIs resistance but several studies have already proposed the mechanisms involved and potential interventions. Interestingly, emerging data highlighted that LKB1 alterations represent positive determinants to the new KRAS specific inhibitors response in KRAS co-mutated NSCLCs. In conclusion, the absence of the target did not block the development of treatments able to hit LKB1-mutated NSCLCs acting on several fronts. This will give patients a concrete chance to finally benefit from an effective therapy.

PEGylated recombinant human hyaluronidase (PEGPH20) pre-treatment improves intra-tumour distribution and efficacy of paclitaxel in preclinical models.

BACKGROUND: Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs. METHODS: We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan (hyaluronic acid; HA), one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry. RESULTS: Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model. CONCLUSION: Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.

LKB1 Down-Modulation by miR-17 Identifies Patients With NSCLC Having Worse Prognosis Eligible for Energy-Stress-Based Treatments.

INTRODUCTION: Preclinical models recently unveiled the vulnerability of LKB1/KRAS comutated NSCLC to metabolic stress-based treatments. Because miR-17 is a potential epigenetic regulator of LKB1, we hypothesized that wild-type LKB1 (LKB1WT) NSCLC with high miR-17 expression may be sensitive to an energetic stress condition, and eligible for metabolic frailties-based therapeutic intervention. METHODS: We took advantage of NSCLC cell lines with different combinations of KRAS mutation and LKB1 deletion and of patient-derived xenografts (PDXs) with high (LKB1WT/miR-17 high) or low (LKB1WT/miR-17 low) miR-17 expression. We evaluated LKB1 pathway impairment and apoptotic response to metformin. We retrospectively evaluated LKB1 and miR-17 expression levels in tissue specimens of patients with NSCLC and PDXs. In addition, a lung cancer series from The Cancer Genome Atlas data set was analyzed for miR-17 expression and potential correlation with clinical features. RESULTS: We identified miR-17 as an epigenetic regulator of LKB1 in NSCLC and confirmed targeting of miR-17 to LKB1 3' untranslated region by luciferase reporter assay. We found that miR-17 overexpression functionally impairs the LKB1/AMPK pathway. Metformin treatment prompted apoptosis on miR-17 overexpression only in LKB1WT cell lines, and in LKB1WT/miR-17 high PDXs. A retrospective analysis in patients with NSCLC revealed an inverse correlation between miR-17 and LKB1 expression and highlighted a prognostic role of miR-17 expression in LKB1WT patients, which was further confirmed by The Cancer Genome Atlas data analysis. CONCLUSIONS: We identified miR-17 as a mediator of LKB1 expression in NSCLC tumors. This study proposes a miR-17 expression score potentially exploitable to discriminate LKB1WT patients with NSCLC with impaired LKB1 expression and poor outcome, eligible for energy-stress-based treatments.

Activity of Birinapant, a SMAC Mimetic Compound, Alone or in Combination in NSCLCs With Different Mutations.

Liver kinase B1 (LKB1/STK11) is the second tumor suppressor gene most frequently mutated in non-small-cell lung cancer (NSCLC) and its activity is impaired in about half KRAS-mutated NSCLCs. Nowadays, no effective therapies are available for patients having these mutations. To highlight new vulnerabilities of this subgroup of tumors exploitable to design specific therapies we screened an US FDA-approved drug library using an isogenic system of wild-type (WT) or deleted LKB1. Among eight hit compounds, Birinapant, an inhibitor of the Inhibitor of Apoptosis Proteins (IAPs), was the most active compound in LKB1-deleted clone only compared to its LKB1 WT counterpart. We validated the Birinapant cells response and its mechanism of action to be dependent on LKB1 deletion. Indeed, we demonstrated the ability of this compound to induce apoptosis, through activation of caspases in the LKB1-deleted clone only. Expanding our results, we found that the presence of KRAS mutations could mediate Birinapant resistance in a panel of NSCLC cell lines. The combination of Birinapant with Ralimetinib, inhibitor of p38α, restores the sensitivity of LKB1- and KRAS-mutated cell lines to the IAP inhibitor Birinapant. Our study shows how the use of Birinapant could be a viable therapeutic option for patients with LKB1-mutated NSCLCs. In addition, combination of Birinapant and a KRAS pathway inhibitor, as Ralimetinib, could be useful for patients with LKB1 and KRAS-mutated NSCLC.

Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation.

Non-small-cell lung cancer (NSCLC) cell lines vary in their sensitivity to glutaminase inhibitors, so it is important to identify the metabolic assets underling their efficacy in cancer cells. Even though specific genetic lesions such as in KRAS and LKB1 have been associated with reliance on glutamine for their metabolic needs, we found no distinction between glutaminase inhibitor CB-839 sensitivity and resistant phenotypes in NSCLC cells with or without these genetic alterations. We demonstrated the close relationship between environmental alanine uptake and catabolism. This response depended on the individual cell's ability to employ alanine aminotransferase (GPT2) to compensate the reduced glutamate availability. It may, therefore, be useful to determine GPT2 levels to predict which NSCLC patients would benefit most from glutaminase inhibitor treatment.

Molecular determinants of response to PI3K/akt/mTOR and KRAS pathways inhibitors in NSCLC cell lines.

Despite the impressive results obtained in the preclinical setting, all the inhibitors targeting two central cascades in cancer, the PI3K/akt/mTOR and the KRAS/MEK/ERK pathways, have shown, apart from very few exceptions, disappointing efficacy when translated to the clinic. One of the main reasons of their clinical failure seems to be the lack of a clear molecular determinant of response to these drugs. In this study, we tried to address this point by evaluating the cytotoxic activity of different inhibitors targeting the two pathways at different levels in a panel of ten NSCLC cell lines harboring alterations in PI3K, KRAS or both. We were not able to highlight a correlation between the presence of KRAS and PI3K mutations and a specific sensitivity to the different drugs used. Molecular analyses performed after equimolar treatments showed that, independently from the entity of the response, the drugs are able to modulate the activation of their targets. Interestingly, we found that p53 mutational status separates the cell lines according to their sensitivity to PI3K pathway inhibitors treatments. The alterations considered in the PI3K/akt/mTOR and in the KRAS/MEK/ERK pathways in the different NSCLC cell lines are not sufficient to drive treatment choice but rather p53 status is a potential biomarker for the activity of this class of drugs.

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

INTRODUCTION: Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy. METHODS: Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo. RESULTS: In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. CONCLUSIONS: This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.