Distribution and Detectability of EGFR Exon 20 Insertion Variants in NSCLC.

INTRODUCTION: EGFR exon 20 insertion (ex20ins) mutations represent 5% to 10% of EGFR mutations in NSCLC. Identifying patients with EGFR ex20ins is challenging owing to the limited coverage of polymerase chain reaction (PCR) assays and the relatively recent use of next-generation sequencing (NGS). This study analyzes the spectrum of EGFR ex20ins variants in a large patient population from a global clinical trial and several real-world cohorts and the ability of PCR kits to identify these alterations. METHODS: We conducted this retrospective analysis in patients with NSCLC who underwent NGS or other sequencing testing and had a known EGFR ex20ins mutation. Patients were gathered from a clinical trial (NCT02716116), a chart review study in Germany, and the LC-SCRUM-Japan, GENIE, and U.S. COTA databases. Proportions of patients with ex20ins variants that could have been detected by six commercially available and widely used PCR kits were calculated in each data set. RESULTS: Overall, 636 patients with NSCLC harboring EGFR ex20ins mutations were included in this analysis and 104 unique EGFR ex20ins variants were identified across the data sources. The proportion of patients whose ex20ins could have been detected by any PCR test alone ranged from 11.8% to 58.9% across the data sources. CONCLUSIONS: Our findings suggest that the PCR tests evaluated would have missed more than 40% of patients with NSCLC harboring EGFR ex20ins mutations. NGS-based genetic testing is preferable than standard PCR assays and can substantially improve the identification of the diverse profile of EGFR ex20ins variants in NSCLC.

Non-small cell lung cancer with EGFR exon 20 insertion mutation: a systematic literature review and meta-analysis of patient outcomes.

INTRODUCTION: EGFR exon 20 insertion mutation-positive non-small cell lung cancer (NSCLC) is rare, has a poor prognosis, and outcomes are not fully established. We describe and evaluate outcomes from real-world and clinical evidence in these patients. METHODS: A systematic literature review (SLR) identified interventional and real-world evidence (RWE) studies reporting clinical outcomes for EGFR exon 20 insertion mutation-positive NSCLC. Meta-analyses were conducted by line of therapy to synthesize pooled survival and response outcomes across RWE. Published evidence from interventional studies was summarized individually. RESULTS: The SLR identified 23 RWE and 19 original interventional studies. In the meta-analysis of RWE, pooled response and survival outcomes were low for first-line EGFR-tyrosine kinase inhibitors (TKIs) and immuno-oncology (IO) agents. First-line chemotherapy resulted in a pooled ORR 25.7%, pooled PFS 5.6 months, and pooled OS 18.3 months. Pooled outcomes were further reduced in second or later lines (≥2 L): pooled ORR was 5.0%, 3.3%, and 13.9%; pooled PFS was 2.1 months, 2.3 months, and 4.4 months; and pooled OS was 14.1 months, 8.8 months, and 17.1 months (not a pooled result) for EGFR-TKIs, IO agents, and chemotherapy, respectively. Interventional studies reported outcomes for TKIs (mobocertinib, poziotinib, osimertinib, afatinib, CLN-081, DZD9008), a monoclonal antibody (amivantamab), and a heat shock protein 90 inhibitor (luminespib). While there is limited RWE for the recently approved agents mobocertinib and amivantamab, which specifically target exon 20 insertion mutations, interventional evidence supports their potential as effective treatment options. CONCLUSIONS: Conventional treatments used in patients with EGFR exon 20 insertion mutation-positive NSCLC have limited efficacy, though chemotherapy appeared to be associated with better response and survival outcomes than non-exon 20 targeting EGFR-TKIs and IO agents. This supports the need to identify EGFR exon 20 insertion mutations as the availability of new targeted treatments may offer additional therapeutic options to these patients.

Pharmacological inhibition of ßIIPKC is cardioprotective in late-stage hypertrophy.

We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, ßIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a ßIIPKC-specific inhibitor, ßIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the ßΙΙPKC-treated rats, suggesting that ßΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of ßIPKC, an alternative spliced form of ßIIPKC, had no beneficial effects in these rats. We also found that ßIIV5-3, but not ßIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that ßIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of ßIIPKC may be a potential treatment for this disease.

Rationally designed peptide regulators of protein kinase C.

Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Whereas the earlier inhibitory peptides are highly specific, we believe that peptides targeting additional interactions between PKC and selective substrates will generate even more selective tools that regulate different functions of individual isozymes. Here, we discuss the methodologies and applications for identifying selective regulators of PKC.

Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2.

The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (varepsilonPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of varepsilonPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of varepsilonPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and varepsilonPKC-mediated protection from cardiac ischemia, in vivo.

Reperfusion-induced translocation of deltaPKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation.

Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive delta-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the deltaPKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of deltaPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of deltaPKC is associated with phosphorylation of the alphaE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that deltaPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of deltaPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

Mitigation of radiation-induced dermatitis by activation of aldehyde dehydrogenase 2 using topical alda-1 in mice.

Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy.

Ischaemic preconditioning improves proteasomal activity and increases the degradation of deltaPKC during reperfusion.

AIMS: The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilonPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. METHODS AND RESULTS: Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished deltaPKC translocation by 3.8-fold and increased epsilonPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of deltaPKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilonPKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilonPKC during IPC restored deltaPKC levels at the mitochondria while decreasing epsilonPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a deltaPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. CONCLUSION: Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, deltaPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilonPKC.

Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

AIMS: Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon. METHODS AND RESULTS: Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction. CONCLUSION: Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.

Ethanol for cardiac ischemia: the role of protein kinase c.

The physiological effects of ethanol are dependent upon the amount and duration of consumption. Chronic excessive consumption can lead to diseases such as liver cirrhosis, and cardiac arrhythmias, while chronic moderate consumption can have therapeutic effects on the cardiovascular system. Recently, it has also been observed that acute administration of ethanol to animals prior to an ischemic event provides significant protection to the heart. This review focuses on the different modalities of chronic vs. acute ethanol consumption and discusses recent evidence for a protective effect of acute ethanol exposure and the possible use of ethanol as a therapeutic agent.

Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart.

There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here, using an unbiased proteomic search, we identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats before an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wild-type or mutant ALDH2 who are subjected to cardiac ischemia, such as during coronary bypass surgery.

Cardioprotective mechanisms of PKC isozyme-selective activators and inhibitors in the treatment of ischemia-reperfusion injury.

Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of ischemia by either mechanical (balloon catheters) or enzymatic (thrombolytics) means to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury, which occurs after these interventions. The development of rationally designed PKC isozyme-selective regulator peptides has permitted investigation into the role of specific PKC isozymes in ischemia-reperfusion (IR) injury. Based on these studies, it is now evident that epsilon and deltaPKC have distinct temporal and opposing roles in regulating myocardial damage induced by IR. Activation of epsilonPKC before ischemia protects the heart by mimicking preconditioning, whereas inhibition of deltaPKC during reperfusion protects the heart from reperfusion-induced damage. These cardioprotective effects have been observed in isolated cardiomyocytes, isolated perfused hearts and in vivo in all species tested including mouse, rat and pig and may provide the basis for future therapeutic agents. Having established the efficacy of PKC isozyme-specific regulators in reducing IR injury, the next challenge is to outline the molecular mechanisms regulated by delta and epsilonPKC isozymes that result in enhanced tolerance to IR. In this review, we discuss progress that has been made in establishing cytoprotective mechanisms, which arise as a consequence of epsilonPKC activation or deltaPKC inhibition, and how they may lead to protection in the setting of myocardial ischemia reperfusion.

Translocation of deltaPKC to mitochondria during cardiac reperfusion enhances superoxide anion production and induces loss in mitochondrial function.

Activation of the delta-isoform of protein kinase C (deltaPKC) by certain conditions of oxidative stress results in translocation of the kinase to the mitochondria leading to release of cytochrome c and the induction of apoptosis. In the current study, the effects of myocardial reperfusion-induced deltaPKC translocation on mitochondrial function were assessed. Mitochondria isolated from hearts that had undergone ischemia (30 min) followed by reperfusion (15 min) exhibited a significant increase in the rate of superoxide anion (O(2)(-)) generation. This was associated with the translocation of deltaPKC to the mitochondria within the first 5 min of reperfusion. deltaPKC translocation occurred exclusively during reperfusion and could be mimicked by infusion of intact hearts with H(2)O(2) suggesting redox-dependent activation during reperfusion. Infusion of a peptide inhibitor (deltaV(1-1)) specific to the delta-isoform of PKC significantly reduced reperfusion-induced increases in mitochondrial O(2)(-) generation. Finally, the decline in mitochondrial respiratory activity evident upon prolonged reperfusion (120min) was completely prevented by inhibition of deltaPKC translocation. Thus, deltaPKC represents a cytosolic redox-sensitive molecule that plays an important role in amplification of O(2)(-) production and subsequent declines in mitochondrial function during reperfusion.