DNA topoisomerase II inhibition potentiates osimertinib's therapeutic efficacy in EGFR-mutant non-small cell lung cancer models.

Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.

Taking early preventive interventions to manage the challenging issue of acquired resistance to third-generation EGFR inhibitors.

Although the clinical efficacies of third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as osimertinib in the treatment of non-small cell lung cancer (NSCLC) with EGFR-activating mutations are promising, drug-acquired resistance inevitably occurs whether they are used as first-line or second-line treatment. Therefore, managing the acquired resistance to third-generation EGFR-TKIs is crucial in the clinic for improving patient survival. Great efforts have been made to develop potentially effective strategies or regimens for the treatment of EGFR-mutant NSCLC patients after relapse following these TKIs therapies with the hope that patients will continue to benefit from treatment through overcoming acquired resistance. Although this approach, which aims to overcome drug-acquired resistance, is necessary and important, it is a passive practice. Taking preventive action early before disease progression to manage the unavoidable development of acquired resistance offers an equally important and efficient approach. We strongly believe that early preventive interventions using effective and tolerable combination regimens that interfere with the process of developing acquired resistance may substantially improve the outcomes of EGFR-mutant NSCLC treatment with third-generation EGFR-TKIs. Thus, this review focuses on discussing the scientific rationale and mechanism-driven strategies for delaying and even preventing the emergence of acquired resistance to third-generation EGFR-TKIs, particularly osimertinib.

ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant lung cancer cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression.

The successful treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) with ALK tyrosine kinase inhibitors (ALK-TKIs) represents a promising targeted therapy. As a result, various ALK-TKIs have been rapidly developed, some of which are approved while some are being tested in clinical trials. Death receptor 4 (DR4; also called TNFRSF10A or TRAIL-R1) is a cell surface protein, which functions as a pro-apoptotic protein that transduces TRAIL death signaling to trigger apoptosis. DR4 expression is positively regulated by MEK/ERK signaling and thus can be downregulated by MEK/ERK inhibition. This study thus focused on determining the effects of AKL-TKIs on DR4 expression and the underlying mechanisms. Three tested ALK-TKIs including APG-2449, brigatinib and alectinib effectively and preferentially inhibited Akt/mTOR as well as MEK/ERK signaling and decreased cell survival in ALK-mutant (ALKm) NSCLC cells with induction of apoptosis. This was also true for DR4 downregulation, which occurred even at 2 h post treatment. These ALK-TKIs did not affect DR4 protein stability, rather decreased DR4 mRNA expression. In parallel, they promoted degradation and reduced the levels of Fra-1 and c-Jun, two critical components of AP-1, and suppressed AP-1 (Fra-1/c-Jun)-dependent transcription/expression of DR4. Hence, it appears that ALK-TKIs downregulate DR4 expression in ALKm NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. Our findings thus warrant further investigation of the biological significance of DR4 downregulation in ALK-targeted cancer therapy.

Therapeutic potential of the novel Bcl-2/Bcl-XL dual inhibitor, APG1252, alone or in combination against non-small cell lung cancer.

Targeting the induction of apoptosis is a promising cancer therapeutic strategy with some clinical success. This study focused on evaluating the therapeutic efficacy of the novel Bcl-2/Bcl-XL dual inhibitor, APG1252-M1 (also named APG-1244; an in vivo active metabolite of APG1252 or pelcitoclax), as a single agent or in combination, against non-small cell lung cancer (NSCLC) cells. APG1252-M1 effectively decreased the survival of some NSCLC cell lines expressing low levels of Mcl-1 and induced apoptosis. Overexpression of ectopic Mcl-1 in the sensitive cells substantially compromised APG1252-M1's cell-killing effects, whereas inhibition of Mcl-1 greatly sensitized insensitive cell lines to APG1252-M1, indicating the critical role of Mcl-1 levels in impacting cell response to APG1252-M1. Moreover, APG1252-M1, when combined with the third generation epidermal growth factor receptor (EGFR) inhibitor, osimertinib, synergistically decreased the survival of EGFR-mutant NSCLC cell lines including those resistant to osimertinib with enhanced induction of apoptosis and abrogated emergence of acquired resistance to osimertinib. Importantly, the combination was effective in inhibiting the growth of osimertinib-resistant tumors in vivo. Collectively, these results demonstrate the efficacy of APG1252 alone or in combination against human NSCLC cells.

Targeting apoptosis to manage acquired resistance to third generation EGFR inhibitors.

A significant clinical challenge in lung cancer treatment is management of the inevitable acquired resistance to third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs), such as osimertinib, which have shown remarkable success in the treatment of advanced NSCLC with EGFR activating mutations, in order to achieve maximal response duration or treatment remission. Apoptosis is a major type of programmed cell death tightly associated with cancer development and treatment. Evasion of apoptosis is considered a key hallmark of cancer and acquisition of apoptosis resistance is accordingly a key mechanism of drug acquired resistance in cancer therapy. It has been clearly shown that effective induction of apoptosis is a key mechanism for third generation EGFR-TKIs, particularly osimertinib, to exert their therapeutic efficacies and the development of resistance to apoptosis is tightly associated with the emergence of acquired resistance. Hence, restoration of cell sensitivity to undergo apoptosis using various means promises an effective strategy for the management of acquired resistance to third generation EGFR-TKIs.

Multiplexed protein profiling reveals spatial subcellular signaling networks.

Protein-protein interaction networks are altered in multi-gene dysregulations in many disorders. Image-based protein multiplexing sheds light on signaling pathways to dissect cell-to-cell heterogeneity, previously masked by the bulk assays. Herein, we present a rapid multiplexed immunofluorescence (RapMIF) method measuring up to 25-plex spatial protein maps from cultures and tissues at subcellular resolution, providing combinatorial 272 pairwise and 1,360 tri-protein signaling states across 33 multiplexed pixel-level clusters. The RapMIF pipeline automated staining, bleaching, and imaging of the biospecimens in a single platform. RapMIF showed that WNT/ß-catenin signaling upregulated upon the inhibition of the AKT/mTOR pathway. Subcellular protein images demonstrated translocation patterns, spatial receptor discontinuity, and subcellular signaling clusters in single cells. Signaling networks exhibited spatial redistribution of signaling proteins in drug-responsive cultures. Machine learning analysis predicted the phosphorylated ß-catenin expression from interconnected signaling protein images. RapMIF is an ideal signaling discovery approach for precision therapy design.

Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL dual inhibitor that induces Bax-dependent apoptosis.

New treatment options, such as targeted therapies, are urgently needed for the treatment of colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide. The current study focuses on demonstrating the therapeutic efficacies of APG-1252-M1 (an active form of the prodrug, APG-1252 or pelcitoclax), a highly potent Bcl-2/Bcl-XL dual inhibitor in clinical trials, against CRC and understanding the underlying mechanisms. APG-1252-M1 effectively decreased the survival of CRC cell lines, particularly those expressing relatively low levels of Mcl-1, with the induction of apoptosis. High levels of Mcl-1 were significantly correlated with decreased sensitivity of CRC cell lines to APG-1252-M1. When combined with an Mcl-1 inhibitor, APG-1252-M1 synergistically decreased the survival and induced apoptosis of APG-1252-M1-insensitive cell lines with high levels of Mcl-1. This combination further decreased the survival and enhanced apoptosis even in sensitive cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG-1252-M1's effects on decreasing cell survival and inducing apoptosis, which could be reversed by Mcl-1 inhibition. APG-1252-M1 rapidly induced cytochrome C and Smac release from mitochondria with caspase-3 and PARP cleavage. Deficiency of Bax in CRC cells abolished APG-1252-M1's ability to induce apoptosis, indicating that APG-1252-M1 induces Bax-dependent apoptosis. The current study thus demonstrates the potential of APG-1252-M1 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with an Mcl-1 inhibitor, warranting further evaluation in vivo and in the clinic.

Therapeutic efficacy of the novel SHP2 degrader SHP2-D26, alone or in combination, against lung cancer is associated with modulation of p70S6K/S6, Bim and Mcl-1.

SHP2, a protein tyrosine phosphatase, plays a critical role in fully activating oncogenic signaling pathways such as Ras/MAPK downstream of cell surface tyrosine receptors (e.g., EGFR), which are often activated in human cancers, and thus has emerged as an attractive cancer therapeutic target. This study focused on evaluating the therapeutic potential of the novel SHP2 degrader, SHP2-D26 (D26), either alone or in combination, against non-small cell lung cancer (NSCLC) cells. While all tested NSCLC cell lines responded to D26 with IC50s of < 8 µM, a few cell lines (4/14) were much more sensitive than others with IC50s of ≤ 4 µM. There was no clear association between basal levels of SHP2 and cell sensitivities to D26. Moreover, D26 rapidly and potently decreased SHP2 levels in different NSCLC cell lines in a sustained way regardless of cell sensitivities to D26, suggesting that additional factors may impact cell response to D26. We noted that suppression of p70S6K/S6, but not ERK1/2, was associated with cell responses to D26. In the sensitive cell lines, D26 effectively increased Bim levels while decreasing Mcl-1 levels accompanied with the induction of apoptosis. When combined with the third generation EGFR inhibitor, osimertinib (AZD9291), synergistic effects on decreasing the survival of different osimertinib-resistant cell lines were observed with enhanced induction of apoptosis. Although D26 alone exerted moderate inhibition of the growth of NSCLC xenografts, the combination of osimertinib and D26 effectively inhibited the growth of osimertinib-resistant xenografts, suggesting promising efficacy in overcoming acquired resistance to osimertinib.

The novel BET degrader, QCA570, is highly active against the growth of human NSCLC cells and synergizes with osimertinib in suppressing osimertinib-resistant EGFR-mutant NSCLC cells.

Lung cancer remains the leading cause of cancer deaths worldwide despite advances in knowledge in cancer biology and options of various targeted therapies. Efforts in identifying innovative and effective therapies are still highly appreciated. Targeting bromodomain and extra terminal (BET) proteins that function as epigenetic readers and master transcription coactivators is now a potential cancer therapeutic strategy. The current study evaluates the therapeutic efficacies of the novel BET degrader, QCA570, in lung cancer and explores its underlying mechanisms. QCA570 at low nanomolar ranges effectively decreased the survival of a panel of human lung cancer cell lines with induction of apoptosis in vitro. As expected, it potently induced degradation of BET proteins including BRD4, BRD3 and BRD2. Moreover, it potently decreased Mcl-1 levels due to transcriptional suppression and protein degradation; this event is critical for mediating apoptosis induced by QCA570. Moreover, QCA570 synergized with osimertinib in suppressing the growth of osimertinib-resistant cells in vitro and in vivo, suggesting potential in overcoming acquired resistance to osimertinib. These preclinical findings support the potential of QCA570 in treatment of lung cancer either as a single agent or in combination with others.

Overcoming acquired resistance to third-generation EGFR inhibitors by targeting activation of intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation, or both.

Treatment of EGFR-mutant non-small cell lung cancer (NSCLC) with mutation-selective third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib has achieved remarkable success in the clinic. However, the immediate challenge is the emergence of acquired resistance, limiting the long-term remission of patients. This study suggests a novel strategy to overcome acquired resistance to osimertinib and other third-generation EGFR-TKIs through directly targeting the intrinsic apoptotic pathway. We found that osimertinib, when combined with Mcl-1 inhibition or Bax activation, synergistically decreased the survival of different osimertinib-resistant cell lines, enhanced the induction of intrinsic apoptosis, and inhibited the growth of osimertinib-resistant tumor in vivo. Interestingly, the triple-combination of osimertinib with Mcl-1 inhibition and Bax activation exhibited the most potent activity in decreasing the survival and inducing apoptosis of osimertinib-resistant cells and in suppressing the growth of osimertinib-resistant tumors. These effects were associated with increased activation of the intrinsic apoptotic pathway evidenced by augmented mitochondrial cytochrome C and Smac release. Hence, this study convincingly demonstrates a novel strategy for overcoming acquired resistance to osimertinib and other 3rd generation EGFR-TKIs by targeting activation of the intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation or both, warranting further clinical validation of this strategy.

The natural product berberine synergizes with osimertinib preferentially against MET-amplified osimertinib-resistant lung cancer via direct MET inhibition.

Berberine is a natural product that has long been used in traditional Chinese medicine due to its antimicrobial, anti-inflammatory and metabolism-regulatory properties. Osimertinib is the first third-generation EGFR-tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations and those resistant to earlier generation EGFR-TKIs due to a T790M mutation. However, emergence of acquired resistance to osimertinib limits its long-term efficacy in the clinic. One known mechanism of acquired resistance to osimertinib and other EGFR-TKIs is MET (c-MET) gene amplification. Here, we report that berberine, when combined with osimertinib, synergistically and selectively decreased the survival of several MET-amplified osimertinib-resistant EGFR mutant NSCLC cell lines with enhanced induction of apoptosis likely through Bim elevation and Mcl-1 reduction. Importantly, this combination effectively enhanced suppressive effect on the growth of MET-amplified osimertinib-resistant xenografts in nude mice and was well tolerated. Molecular modeling showed that berberine was able to bind to the kinase domain of non-phosphorylated MET, occupy the front of the binding pocket, and interact with the activation loop, in a similar way as other known MET inhibitors do. MET kinase assay showed clear concentration-dependent inhibitory effects of berberine against MET activity, confirming its kinase inhibitory activity. These findings collectively suggest that berberine can act as a naturally-existing MET inhibitor to synergize with osimertinib in overcoming osimertinib acquired resistance caused by MET amplification.

Pan-cancer analysis of pathway-based gene expression pattern at the individual level reveals biomarkers of clinical prognosis.

Identifying biomarkers to predict the clinical outcomes of individual patients is a fundamental problem in clinical oncology. Multiple single-gene biomarkers have already been identified and used in clinics. However, multiple oncogenes or tumor-suppressor genes are involved during the process of tumorigenesis. Additionally, the efficacy of single-gene biomarkers is limited by the extensively variable expression levels measured by high-throughput assays. In this study, we hypothesize that in individual tumor samples, the disruption of transcription homeostasis in key pathways or gene sets plays an important role in tumorigenesis and has profound implications for the patient's clinical outcome. We devised a computational method named iPath to identify, at the individual-sample level, which pathways or gene sets significantly deviate from their norms. We conducted a pan-cancer analysis and demonstrated that iPath is capable of identifying highly predictive biomarkers for clinical outcomes, including overall survival, tumor subtypes, and tumor-stage classifications.

Induction of SREBP1 degradation coupled with suppression of SREBP1-mediated lipogenesis impacts the response of EGFR mutant NSCLC cells to osimertinib.

Emergence of acquired resistance to osimertinib (AZD9291), the first-approved third-generation EGFR inhibitor that selectively and irreversibly inhibits the activating EGFR mutations and the resistant T790M mutation, is a giant and urgent clinical challenge. Fully understanding the biology underlying the response of EGFR mutant non-small cell lung cancer (NSCLC) to osimertinib is the foundation for development of mechanism-driven strategies to overcome acquired resistance to osimertinib or other third-generation EGFR inhibitors. This study focused on tackling this important issue by elucidating the critical role of sterol regulatory element-binding protein 1 (SREBP1) degradation in conferring the response of EGFR mutant NSCLC cells to osimertinib and by validating the strategy via directly targeting SREBP1 for overcoming osimertinib acquired resistance. Osimertinib facilitated degradation of the mature form of SREBP1 (mSREBP1) in a GSK3/FBXW7-dependent manner and reduced protein levels of its regulated genes in EGFR-mutant NSCLC cells/tumors accompanied with suppression of lipogenesis. Once resistant, EGFR-mutant NSCLC cell lines possessed elevated levels of mSREBP1, which were resistant to osimertinib modulation. Both genetic and pharmacological inhibition of SREBP1 sensitized osimertinib-resistant cells and tumors to osimertinib primarily through enhancing Bim-dependent induction of apoptosis, whereas enforced expression of ectopic SREBP1 in sensitive EGFR-mutant NSCLC cells compromised osimertinib's cell-killing effects. Collectively, we have demonstrated a novel connection between osimertinib and SREBP1 degradation and its impact on the response of EGFR mutant NSCLC cells to osimertinib and suggested an effective strategy for overcoming acquired resistance to osimertinib, and possibly other EGFR inhibitors, via targeting SREBP1.

Inhibition of MEK5/ERK5 signaling overcomes acquired resistance to the third generation EGFR inhibitor, osimertinib, via enhancing Bim-dependent apoptosis.

The promising therapeutic efficacy of the third generation EGFR inhibitor, osimertinib (AZD9291), for the treatment of patients with EGFR-mutant non-small cell lung cancer (NSCLC) has been demonstrated in the clinic both as first-line and second line therapy. However, inevitable acquired resistance limits its long-term benefit to patients and is thus a significant clinical challenge. The current study focuses on studying the potential role of targeting MEK5-ERK5 signaling in overcoming acquired resistance to osimertinib. Osimertinib and other third generation EGFR inhibitors exerted a rapid and sustained suppressive effect on ERK5 phosphorylation primarily in EGFR-mutant NSCLC cell lines and lost this activity in some osimertinib-resistant cell lines. Osimertinib combined with either ERK5 or MEK5 inhibitors synergistically decreased the survival of osimertinib-resistant cell lines with enhanced induction of apoptosis primarily via augmenting Bim expression. Moreover, the combination effectively inhibited the growth of osimertinib-resistant xenografts in vivo. Together, these findings suggest the potential role of MEK5-ERK5 signaling in modulating development of acquired resistance to osimertinib and value of targeting this signaling as a potential strategy in overcoming acquired resistance to osimertinib and possibly other third generation EGFR inhibitors.

MET inhibition downregulates DR4 expression in MET-amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription.

Death receptor 4 (DR4) is a cell surface protein that is generally thought to mediate apoptosis upon binding to its ligand named TRAIL. However, its contribution to apoptosis resistance has also been reported. MET (or c-MET) gene amplification represents an important mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) against EGFR mutant non-small cell lung cancer (NSCLC). This study focuses on demonstrating the impact of MET inhibition on DR4 modulation in MET-amplified EGFR mutant NSCLC cell lines and the underlying mechanisms. Several MET inhibitors decreased DR4 levels in MET-amplified HCC827 cell lines resistant to EGFR-TKIs with no or limited effects on modulating DR5 levels, while increasing DR4 levels in HCC827 parental cells and other NSCLC cell lines. MET inhibitors did not affect DR4 stability, but decreased DR4 mRNA levels with suppression of AP-1-dependent DR4 promoter transactivation. Moreover, these inhibitors suppressed ERK and c-Jun phosphorylation accompanied with decreasing c-Jun levels. Hence, it is likely that MET inhibition downregulates DR4 expression in MET-amplified EGFR mutant NSCLC cells through suppressing AP-1-mediated DR4 transcription. Osimertinib combined with MET inhibition synergistically induces apoptosis in the MET-amplified EGFR mutant NSCLC cells accompanied with augmented DR4 reduction both in vitro and in vivo. Furthermore, MET inhibition combined with TRAIL enhanced killing of MET-amplified EGFR mutant HCC827/AR cells, but not HCC827 parental cells. These data collectively suggest that DR4 may possess an unrecognized anti-apoptotic function, contributing to apoptosis resistance under given conditions.

Targeting c-Myc to Overcome Acquired Resistance of EGFR Mutant NSCLC Cells to the Third-Generation EGFR Tyrosine Kinase Inhibitor, Osimertinib.

Osimertinib (AZD9291 or TAGRISSO) is a promising and approved third-generation EGFR tyrosine kinase inhibitor (TKI) for treating patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, the inevitable emergence of acquired resistance limits its long-term efficacy. A fuller understanding of the mechanism of action of osimertinib and its linkage to acquired resistance will enable the development of more efficacious therapeutic strategies. Consequently, we have identified a novel connection between osimertinib or other EGFR-TKIs and c-Myc. Osimertinib rapidly and sustainably decreased c-Myc levels primarily via enhancing protein degradation in EGFR-mutant (EGFRm) NSCLC cell lines and xenograft tumors. c-Myc levels were substantially elevated in different EGFRm NSCLC cell lines with acquired resistance to osimertinib in comparison with their corresponding parental cell lines and could not be reduced any further by osimertinib. Consistently, c-Myc levels were elevated in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment compared with their corresponding untreated baseline c-Myc levels. Suppression of c-Myc through knockdown or pharmacologic targeting with BET inhibitors restored the response of resistant cell lines to osimertinib. These findings indicate that c-Myc modulation mediates the therapeutic efficacy of osimertinib and the development of osimertinib acquired resistance. Furthermore, they establish c-Myc as a potential therapeutic target and warrant clinical testing of BET inhibition as a potential strategy to overcome acquired resistance to osimertinib or other EGFR inhibitors. SIGNIFICANCE: This study demonstrates a critical role of c-Myc modulation in mediating therapeutic efficacy of osimertinib including osimertinib acquired resistance and suggests targeting c-Myc as a potential strategy to overcome osimertinib acquired resistance.