FDA Approval Summary: Mobocertinib for Metastatic Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations.

On September 15, 2021, the FDA granted accelerated approval to mobocertinib (Exkivity, Takeda Pharmaceuticals USA, Inc.) for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations, as detected by an FDA-approved test, whose disease has progressed on or after platinum-based chemotherapy. The approval was based on data from Study AP32788-15-101 (NCT02716116), an international, non-randomized, multi-cohort clinical trial that included patients with locally advanced or metastatic NSCLC with EGFR exon 20 insertion mutations. The overall response rate in 114 patients whose disease had progressed on or after platinum-based chemotherapy was 28% [95% confidence interval (CI), 20%-37%] with a median duration of response of 17.5 months (95% CI, 7.4-20.3). The most common adverse reactions (>20%) were diarrhea, rash, nausea, stomatitis, vomiting, decreased appetite, paronychia, fatigue, dry skin, and musculoskeletal pain. Product labeling includes a Boxed Warning for QTc prolongation and torsades de pointes. This is the first approval of an oral targeted therapy for patients with advanced EGFR exon 20 insertion mutation-positive NSCLC.

FDA Approval Summary: Capmatinib and Tepotinib for the Treatment of Metastatic NSCLC Harboring MET Exon 14 Skipping Mutations or Alterations.

The FDA approved capmatinib and tepotinib on May 6, 2020, and February 3, 2021, respectively. Capmatinib is indicated for patients with metastatic non-small cell lung cancer (mNSCLC) whose tumors have a mutation leading to mesenchymal-epithelial transition (MET) exon 14 skipping as detected by an FDA-approved test. Tepotinib is indicated for mNSCLC harboring MET exon 14 skipping alterations. The approvals were based on trials GEOMETRY mono-1 (capmatinib) and VISION (tepotinib). In GEOMETRY mono-1, overall response rate (ORR) per Blinded Independent Review Committee (BIRC) was 68% [95% confidence interval (CI), 48-84] with median duration of response (DoR) 12.6 months (95% CI, 5.5-25.3) in 28 treatment-naïve patients and 41% (95% CI: 29, 53) with median DoR 9.7 months (95% CI, 5.5-13) in 69 previously treated patients with NSCLC with mutations leading to MET exon 14 skipping. In VISION, ORR per BIRC was 43% (95% CI: 32, 56) with median DoR 10.8 months (95% CI, 6.9-not estimable) in 69 treatment-naïve patients and 43% (95% CI, 33-55) with median DoR 11.1 months (95% CI, 9.5-18.5) in 83 previously-treated patients with NSCLC harboring MET exon 14 alterations. These are the first two therapies to be FDA approved specifically for patients with metastatic NSCLC with MET exon 14 skipping.

FDA Supplemental Approval Summary: Lenvatinib for the Treatment of Unresectable Hepatocellular Carcinoma.

On August 16, 2018, the U.S. Food and Drug Administration approved lenvatinib (Lenvima, Eisai Inc.) for first-line treatment of patients with unresectable hepatocellular carcinoma (HCC). Approval was based on an international, multicenter, randomized, open-label, noninferiority trial (REFLECT; NCT01761266) conducted in 954 patients with previously untreated metastatic or unresectable HCC. Patients were randomized (1:1) to receive lenvatinib (12 mg orally once daily for patients with a baseline body weight ≥60 kg and 8 mg orally once daily for patients with a baseline body weight <60 kg) or sorafenib (400 mg orally twice daily) until radiological disease progression or unacceptable toxicity. REFLECT demonstrated that lenvatinib was noninferior but not statistically superior to sorafenib for overall survival (OS; hazard ratio, [HR] 0.92; 95% confidence intervals [CI], 0.79-1.06), with median OS of 13.6 and 12.3 months in the lenvatinib and sorafenib arms, respectively. REFLECT also demonstrated statistically significant improvements in investigator-assessed progression-free survival (PFS; HR, 0.66; 95% CI, 0.57-0.77]; p < 0.001), corresponding to median PFS of 7.4 and 3.7 months and overall response rate of 24.1% vs 9.2% per modified RECIST for HCC (mRECIST) in the lenvatinib and sorafenib arms, respectively. Consistent results were observed by an independent review facility per RECISTv1.1 and per mRECIST. The most common adverse reactions observed in the lenvatinib-treated patients (≥20%) in decreasing frequency were hypertension, fatigue, diarrhea, decreased appetite, arthralgia/myalgia, decreased weight, abdominal pain, palmar-plantar erythrodysesthesia syndrome, proteinuria, dysphonia, hemorrhagic events, hypothyroidism, and nausea. IMPLICATIONS FOR PRACTICE: This article describes the U.S. Food and Drug Administration's review of data from a single trial, REFLECT, that supported the approval of lenvatinib, as a single agent, for the first-line treatment of unresectable hepatocellular carcinoma (HCC). REFLECT was an open-label, noninferiority trial that randomized 954 patients with HCC who were ineligible for liver-directed therapy with no prior systemic therapy for HCC to lenvatinib or sorafenib. REFLECT demonstrated that lenvatinib-treated patients had similar survival, more responses, and longer time to progression than those receiving sorafenib. Serious side effects were more common among lenvatinib-treated patients. Lenvatinib is an effective treatment for patients with previously untreated HCC.

Differential Expression of OATP1B3 Mediates Unconjugated Testosterone Influx.

Castration-resistant prostate cancer (CRPC) has greater intratumoral testosterone concentrations than similar tumors from eugonadal men; simple diffusion does not account for this observation. This study was undertaken to ascertain the androgen uptake kinetics, functional, and clinical relevance of de novo expression of the steroid hormone transporter OATP1B3 (SLCO1B3). Experiments testing the cellular uptake of androgens suggest that testosterone is an excellent substrate of OATP1B3 (Km = 23.2 µmol/L; Vmax = 321.6 pmol/mg/minute), and cells expressing a doxycycline-inducible SLCO1B3 construct had greater uptake of a clinically relevant concentration of 3H-testosterone (50 nmol/L; 1.6-fold, P = 0.0027). When compared with Slco1b2 (-/-) mice, Slco1b2 (-/-)/hSLCO1B3 knockins had greater hepatic uptake (15% greater AUC, P = 0.0040) and lower plasma exposure to 3H-testosterone (17% lower AUC, P = 0.0030). Of 82 transporters genes, SLCO1B3 is the second-most differentially expressed transporter in CRPC cell lines (116-fold vs. androgen-sensitive cells), with a differentially spliced cancer-type ct-SLCO1B3 making up the majority of SLCO1B3 expression. Overexpression of SLCO1B3 in androgen-responsive cells results in 1.5- to 2-fold greater testosterone uptake, whereas siRNA knockdown of SLCO1B3 in CRPC cells did not change intracellular testosterone concentration. Primary human prostate tumors express SLCO1B3 to a greater extent than ct-SLCO1B3 (26% of total SLCO1B3 expression vs. 0.08%), suggesting that androgen uptake in these tumor cells also is greater. Non-liver tumors do not differentially express SLCO1B3.Implications: This study suggests that de novo OATP1B3 expression in prostate cancer drives greater androgen uptake and is consistent with previous observations that greater OATP1B3 activity results in the development of androgen deprivation therapy resistance and shorter overall survival. Mol Cancer Res; 15(8); 1096-105. ©2017 AACR.

Dual targeting of the androgen receptor and hypoxia-inducible factor 1α pathways synergistically inhibits castration-resistant prostate cancer cells.

Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). Although enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and hypoxia inducible factor-1 α (HIF-1α) are key regulators of these processes, dual targeting of both signaling axes represents an attractive therapeutic approach. Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. In prostate cancer cells, the gene expression of AR targets (KLK3, FKBP5, TMPRSS2) was repressed by HIF-signaling; conversely, specific HIF-1α target expression was induced by dihydrotestosterone-mediated AR signaling. Treatment of CRPC cells with enzalutamide or HIF-1α inhibition attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced vascular endothelial growth factor protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells via increased enzalutamide-induced apoptosis. In conclusion, the combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene-specific HIF-dependent expression and prostate cancer cell growth.

Inhibition of the HIF1α-p300 interaction by quinone- and indandione-mediated ejection of structural Zn(II).

Protein-protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds.

Visualization of subunit interactions and ternary complexes of protein phosphatase 2A in mammalian cells.

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.

Epidithiodiketopiperazines (ETPs) exhibit in vitro antiangiogenic and in vivo antitumor activity by disrupting the HIF-1α/p300 complex in a preclinical model of prostate cancer.

The downstream targets of hypoxia inducible factor-1 alpha (HIF-1α) play an important role in tumor progression and angiogenesis. Therefore, inhibition of HIF-mediated transcription has potential in the treatment of cancer. One attractive strategy for inhibiting HIF activity is the disruption of the HIF-1α/p300 complex, as p300 is a crucial coactivator of hypoxia-inducible transcription. Several members of the epidithiodiketopiperazine (ETP) family of natural products have been shown to disrupt the HIF-1α/p300 complex in vitro; namely, gliotoxin, chaetocin, and chetomin. Here, we further characterized the molecular mechanisms underlying the antiangiogenic and antitumor effects of these ETPs using a preclinical model of prostate cancer. In the rat aortic ring angiogenesis assay, gliotoxin, chaetocin, and chetomin significantly inhibited microvessel outgrowth at a GI50 of 151, 8, and 20 nM, respectively. In vitro co-immunoprecipitation studies in prostate cancer cell extracts demonstrated that these compounds disrupted the HIF-1α/p300 complex. The downstream effects of inhibiting the HIF-1α/p300 interaction were evaluated by determining HIF-1α target gene expression at the mRNA and protein levels. Dose-dependent decreases in levels of secreted VEGF were detected by ELISA in the culture media of treated cells, and the subsequent downregulation of VEGFA, LDHA, and ENO1 HIF-1α target genes were confirmed by semi-quantitative real-time PCR. Finally, treatment with ETPs in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. These results suggest that directly targeting the HIF-1α/p300 complex with ETPs may be an effective approach for inhibiting angiogenesis and tumor growth.

Are race-specific ERCC1 haplotypes in melanoma cases versus controls related to the predictive and prognostic value of ERCC1 N118N?

OBJECTIVES: Although it does not alter the ERCC1 phenotype, the ERCC1 500C>T (rs11615) polymorphism has undergone a myriad of investigations into its role as a marker for nucleotide excision repair (NER) function in different races, diseases and treatment outcomes. The goal of our study was to test the hypothesis that 500C>T is in linkage disequilibrium (LD) with causative alleles, and that these haplotypes are more frequent in Caucasians with melanoma than in healthy Caucasians or African Americans. DESIGN: In this case-control study, we selected race-specific ERCC1 single-nucleotide polymorphism (SNPs), conducted LD analysis with ERCC1 500C>T and compared the frequency of ERCC1 diplotypes in Caucasians with melanoma (n=165), healthy Caucasians (n=150) and healthy African Americans (n=159). The haplotype was further studied using a fusion gene containing multiple ERCC1 SNPs. SETTING: Large cancer institute in the USA. PARTICIPANTS: A total of 165 Caucasian melanoma patients, 159 healthy Caucasian controls and 159 African American healthy controls. Men and women were enrolled in the clinical trial; however, since the screening trial included prostate cancer screening in addition to screening for other cancers, only male controls were available. OUTCOME MEASURES: The outcome measures were melanoma risk in Caucasians, and LD between ERCC1 SNP, N118N and other race-specific allelic variants. RESULTS: When compared to ERCC1 500C>T alone, a race-specific three-SNP variant haplotype in ERCC1 (comprised of rs11615, rs3212950 and rs3212948) was even more frequent in Caucasians with melanoma than in healthy Caucasians (p=0.0034) or African Americans (p<0.0001). A plasmid containing the variant haplotype was not differentially expressed. CONCLUSIONS: We demonstrate that ERCC1 500C>T participates in a previously characterised cancer-risk haplotype found more frequently in Caucasians, while LD is weak in African Americans; this haplotype appears to also be related to melanoma. It is therefore likely that ERCC1 500C>T is only a valid NER, disease or treatment outcome marker in Caucasians.

The ERCC1 N118N polymorphism does not change cellular ERCC1 protein expression or platinum sensitivity.

Genetic polymorphisms in ERCC1 are thought to contribute to altered sensitivity to platinum-based chemotherapy. Although ERCC1 N118N (500 C>T, rs11615) is the most studied polymorphism, the impact of this polymorphism on platinum-based chemotherapy remains unclear. This is the first study in which the functional impact of ERCC1 N118N on gene expression and platinum sensitivity was explored. The aim of this study is to investigate if the reduced codon usage frequency of AAT, which contains the variant allele of the silent mutation, has functional impact on ERCC1 in a well-controlled biological system. Specifically, the ERCC1 cDNA clone with either the C or T allele was introduced into an ERCC1 deficient cell line, UV20, and assayed for the effect of the two alleles on ERCC1 transcription, translation and platinum sensitivity. Both ERCC1 mRNA and protein expression levels increased upon cisplatin treatment, peaking at 4h post-treatment, however there were no differences between the two alleles (p>0.05). Cells complemented with ERCC1 showed significantly higher survival proportion than the parental cell line following platinum exposure (p<0.0001), although no differences were observed between the cells transfected with the wild type or the polymorphic allele. These data suggest that N118N itself is not related to the phenotypic differences in ERCC1 expression or function, but rather this polymorphism may be linked to other causative variants or haplotypes.

The Balpha and Bdelta regulatory subunits of PP2A are necessary for assembly of the CaMKIV.PP2A signaling complex.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Balpha or Bdelta causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV.PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.

Regulation and function of the calcium/calmodulin-dependent protein kinase IV/protein serine/threonine phosphatase 2A signaling complex.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a member of the broad substrate specificity class of Ca(2+)/calmodulin (CaM)-dependent protein kinases and functions as a potent stimulator of Ca(2+)-dependent gene expression. Activation of CaMKIV is a transient, tightly regulated event requiring both Ca(2+)/CaM binding and phosphorylation of the kinase on T200 by an upstream CaMK kinase (CaMKK). Previously, CaMKIV was shown to stably associate with protein serine/threonine phosphatase 2A (PP2A), which was proposed to play a role in negatively regulating the kinase. Here we report that the Ca(2+)/CaM binding-autoinhibitory domain of CaMKIV is required for association of the kinase with PP2A and that binding of PP2A and Ca(2+)/CaM appears to be mutually exclusive. We demonstrate that inhibition of the CaMKIV/PP2A association in cells results in enhanced CaMKIV-mediated gene transcription that is independent of Ca(2+)/CaM. The enhanced transcriptional activity correlates with the elevated level of phospho-T200 that accumulates when CaMKIV is prevented from interacting with PP2A. Collectively, these data suggest a molecular basis for the sequential activation and inactivation of CaMKIV. First, in response to an increase in intracellular Ca(2+), CaMKIV binds Ca(2+)/CaM and becomes phosphorylated on T200 by CaMKK. These events result in the generation of autonomous activity required for CaMKIV-mediated transcriptional regulation. The CaMKIV-associated PP2A then dephosphorylates CaMKIV T200, thereby terminating autonomous activity and CaMKIV-mediated gene transcription.