A single-arm, multicenter, phase II trial of osimertinib in patients with epidermal growth factor receptor exon 18 G719X, exon 20 S768I, or exon 21 L861Q mutations.

BACKGROUND: For patients with stage IV non-small-cell lung cancer with epidermal growth factor receptor (EGFR) exon 19 deletions and exon 21 L858R mutations, osimertinib is the standard of care. Investigating the activity and safety of osimertinib in patients with EGFR exon 18 G719X, exon 20 S768I, or exon 21 L861Q mutations is of clinical interest. PATIENTS AND METHODS: Patients with stage IV non-small-cell lung cancer with confirmed EGFR exon 18 G719X, exon 20 S768I, or exon 21 L861Q mutations were eligible. Patients were required to have measurable disease, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate organ function. Patients were required to be EGFR tyrosine kinase inhibitor-naive. The primary objective was objective response rate, and secondary objectives were progression-free survival, safety, and overall survival. The study used a two-stage design with a plan to enroll 17 patients in the first stage, and the study was terminated after the first stage due to slow accrual. RESULTS: Between May 2018 and March 2020, 17 patients were enrolled and received study therapy. The median age of patients was 70 years (interquartile range 62-76), the majority were female (n = 11), had a performance status of 1 (n = 10), and five patients had brain metastases at baseline. The objective response rate was 47% [95% confidence interval (CI) 23% to 72%], and the radiographic responses observed were partial response (n = 8), stable disease (n = 8), and progressive disease (n = 1). The median progression-free survival was 10.5 months (95% CI 5.0-15.2 months), and the median OS was 13.8 months (95% CI 7.3-29.2 months). The median duration on treatment was 6.1 months (range 3.6-11.9 months), and the most common adverse events (regardless of attribution) were diarrhea, fatigue, anorexia, weight loss, and dyspnea. CONCLUSIONS: This trial suggests osimertinib has activity in patients with these uncommon EGFR mutations.

NF-κB1 p105 suppresses lung tumorigenesis through the Tpl2 kinase but independently of its NF-κB function.

Nuclear factor-κB (NF-κB) is generally believed to be pro-tumorigenic. Here we report a tumor-suppressive function for NF-κB1, the prototypical member of NF-κB. While NF-κB1 downregulation is associated with high lung cancer risk in humans and poor patient survival, NF-κB1-deficient mice are more vulnerable to lung tumorigenesis induced by the smoke carcinogen, urethane. Notably, the tumor-suppressive function of NF-κB1 is independent of its classical role as an NF-κB factor, but instead through stabilization of the Tpl2 kinase. NF-κB1-deficient tumors exhibit 'normal' NF-κB activity, but a decreased protein level of Tpl2. Reconstitution of Tpl2 or the NF-κB1 p105, but not p50 (the processed product of p105), inhibits the tumorigenicity of NF-κB1-deficient lung tumor cells. Remarkably, Tpl2-knockout mice resemble NF-κB1 knockouts in urethane-induced lung tumorigenesis. Mechanistic studies indicate that p105/Tpl2 signaling is required for suppressing urethane-induced lung damage and inflammation, and activating mutations of the K-Ras oncogene. These studies reveal an unexpected, NF-κB-independent but Tpl2-depenednt role of NF-κB1 in lung tumor suppression. These studies also reveal a previously unexplored role of p105/Tpl2 signaling in lung homeostasis.

Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo.

The p53 tumor suppressor plays a key role in the cell's response to genotoxic stress and loss of this 'guardian of the genome' is an important step in carcinogenesis. The ability of p53 to induce apoptosis through transactivation of its target genes is critical for its function as tumor suppressor. We have found that overexpression of p53 in human cancer cell lines resulted in apoptosis as measured by PARP cleavage. Furthermore we observed cleavage of both caspase 9 and caspase 8 after overexpression of p53 and found that p53-dependent apoptosis was inhibited by either cellular (c-Flip-s, Bcl-X(L)) or pharmacological inhibitors of caspase 8 or caspase 9 respectively. These results indicate that p53 is mediating apoptosis through both the mitochondrial and death receptor pathways. To elucidate the relevant p53 target genes and examine the caspase pathways utilized in vivo, we treated p53+/+ and age matched p53-/- mice with 5 Gy ionizing radiation or 0.5 mg/animal dexamethasone and harvested tissues at 0, 6 and 24 h. We examined the mRNA expression of p21, bax, KILLER/DR5, FAS/APO1 and EI24/PIG8 using TaqMan real time quantitative RT-PCR in the spleen, thymus and small intestine. Although the basal mRNA levels of these genes did not depend on the presence of p53, we observed a p53-dependent induction of all these targets in response to gamma-irradiation and a p53-independent regulation for p21 and KILLER/DR5 in response to dexamethasone. Furthermore, we have demonstrated that the relative induction of these p53 target genes is tissue specific. Despite observing otherwise similar levels of death in these tissues, our findings suggest that in some cases apoptosis mediated through p53 occurs by redundant pathways or by a 'group effect' while in other tissues one or few targets may play a key role in p53-dependent apoptosis. Surprisingly, KILLER/DR5 is the dominantly induced transcript in both the spleen and small intestine suggesting a potentially important role for this p53 target gene in vivo.

Identification of inhibitors of TRAIL-induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cell lines, whereas normal cells appear to be protected from its cytotoxic effects. Therefore TRAIL holds promise as a potential therapeutic agent against cancer. To elucidate some of the critical factors that contribute to TRAIL resistance, we performed a genetic screen in the human colon carcinoma cell line SW480 by infecting this TRAIL-sensitive cell line with a human placental cDNA retroviral library and isolating TRAIL-resistant clones. Characterization of the resulting clones for inhibitors of TRAIL-induced death (ITIDs) led to the isolation of c-FLIP(S), Bax inhibitor 1, and Bcl-XL as candidate suppressors of TRAIL signaling. We have demonstrated that c-FLIP(S) and Bcl-XL are sufficient when overexpressed to convey resistance to TRAIL treatment in previously sensitive cell lines. Furthermore both c-FLIP(S) and Bcl-XL protected against overexpression of the TRAIL receptors DR4 and KILLER/DR5. When c-FLIP(S) and Bcl-XL were overexpressed together in SW480 and HCT 116, an additive inhibitory effect was observed after TRAIL treatment suggesting that these two molecules function in the same pathway in the cell lines tested. Furthermore, we have demonstrated for the first time that a proapoptotic member of the Bcl-2 family, Bax, is required for TRAIL-mediated apoptosis in HCT 116 cells. Surprisingly, we have found that the serine/threonine protein kinase Akt, which is an upstream regulator of both c-FLIP(S) and Bcl-XL, is not sufficient when overexpressed to protect against TRAIL in the cell lines tested. These results suggest a key role for c-FLIP(S), Bcl-XL, and Bax in determining tumor cell sensitivity to TRAIL.

The caspase 9 inhibitor Z-LEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis of transformed and cancer cells but not of most normal cells. Recent studies have revealed an unforeseen toxicity of TRAIL toward normal human hepatocytes, thereby bringing into question the safety of systemic administration of TRAIL in humans with cancer. We found that SW480 colon adenocarcinoma, or H460 non-small cell lung cancer cell lines, which are sensitive to TRAIL, were not protected by the caspase 9 inhibitor Z-LEHD-FMK from TRAIL-induced apoptosis. However, a human colon cancer cell line HCT116 and a human embryonic kidney cell line 293, which are sensitive to TRAIL, were protected by Z-LEHD-FMK from TRAIL-mediated death. Both HCT116 and SW480 cells were protected from TRAIL by the caspase 8 inhibitor Z-IETD-FMK, dominant-negative FADD and cellular FLIP-s and interestingly both cell lines displayed caspase 9 cleavage to a similar extent after TRAIL exposure. We confirmed that normal human liver cells are sensitive to TRAIL. Moreover, we found that normal human liver cells could be protected from TRAIL-induced apoptosis by simultaneous exposure to Z-LEHD-FMK. A similar brief exposure to TRAIL plus Z-LEHD-FMK inhibited colony growth of SW480 but not HCT116 cells. Because some cancer cell lines are not protected from TRAIL-mediated killing by Z-LEHD-FMK, we believe that a brief period of caspase 9 inhibition during TRAIL administration may widen the therapeutic window and allow cancer cell killing while protecting normal liver cells. This strategy could be further developed in the effort to advance TRAIL into clinical trials.

Induction of the TRAIL receptor KILLER/DR5 in p53-dependent apoptosis but not growth arrest.

The TRAIL death receptor KILLER/DR5 is induced by DNA damaging agents in wild-type p53-expressing cells. Here we show that, unlike the p53-target CDK-inhibitor p21WAF1/CIP1, the TRAIL death receptor KILLER/DR5 is only induced in cells undergoing p53-dependent apoptosis and not cell cycle arrest. Thus GM glioblastoma cells carrying an inducible MMTV-driven p53 gene undergo cell cycle arrest and upregulate p21 but not KILLER/DR5 expression upon dexamethasone exposure. WI38 normal lung fibroblasts undergoing cell cycle arrest in response to ionizing irradiation also induce p21 but not KILLER/DR5 gene expression. KILLER/DR5 upregulation is also deficient in irradiated lymphoblastoid cells derived from patients with Ataxia Teleangiectasia suggesting a role for the ATM-p53 pathway in regulating KILLER/DR5 expression after DNA damage. Inhibition of transcription by Actinomycin D blocks both KILLER/DR5 and p21 induction in cells undergoing p53-dependent apoptosis. Our results suggest that the p53-dependent transcriptional induction of KILLER/DR5 death receptor is restricted to cells undergoing apoptosis and not cells undergoing exclusively p53-dependent G1 arrest.

BRCA1 signals ARF-dependent stabilization and coactivation of p53.

The hereditary breast and ovarian tumor suppressor BRCA1 can activate p53-dependent gene expression. We show here that BRCA1 increases p53 protein levels through a post-transcriptional mechanism. BRCA1-stabilized p53 has increased sequence-specific DNA-binding and transcriptional activity. BRCA1 does not stabilize p53 in p14ARF-deficient cells. A deletion mutant of BRCA1 which inhibits p53-dependent transcription confers resistance to topoisomerase II-targeted chemotherapy. Our results suggest that BRCA1 may trigger the p53 pathway through two potentially separate mechanisms: accumulation of p53 through a direct or indirect induction of p14ARF as well as direct transcriptional coactivation of p53. BRCA1 may also enhance chemosensitivity and repair of DNA damage through binding to and coactivation of p53.

Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors are members of the tumor necrosis factor superfamily. TRAIL selectively kills cancer cells but not normal cells. We report here the cloning of the mouse homologue of the TRAIL receptor KILLER/DR5 (MK). The cDNA of MK is 1146 bp in length and encodes a protein of 381 amino acids. MK contains an extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic death-domain characteristic of Fas, tumor necrosis factor, and human TRAIL receptors. MK is highly homologous and binds TRAIL with similar affinity as human DR4 and KILLER/DR5. MK induces apoptosis in mouse and human cells and inhibits colony growth of NIH3T3 cells. Expression of MK is p53-dependent and up-regulated by tumor suppressor p53 and by DNA damaging agents in mouse cells undergoing apoptosis. This is the first report describing a mouse TRAIL receptor gene and also demonstrating that the p53-dependent regulation of KILLER/DR5-mediated apoptosis is conserved between human and mouse.

p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha.

The death receptor (DR) KILLER/DR5 gene has recently been identified as a doxorubicin-regulated transcript that was also induced by exogenous wild-type p53 in p53-negative cells. KILLER/DR5 gene encodes a DR containing cell surface protein that is highly homologous to DR4, another DR of the tumor necrosis factor (TNF) receptor family. Both DR4 and KILLER/DR5 independently bind to their specific ligand TRAIL and engage the caspase cascade to induce apoptosis. TRID (also known as TRAIL-R3) is an antiapoptotic decoy receptor that lacks the cytoplasmic death domain and competes with KILLER/DR5 and DR4 for binding to TRAIL. In this study, we demonstrate that the DR KILLER/DR5 gene is regulated in a p53-dependent and -independent manner during genotoxic and nongenotoxic stress-induced apoptosis. Just like other p53-regulated genes, ionizing radiation induction of KILLER/DR5 occurs in p53 wild-type cells, whereas methyl methanesulfonate regulation of KILLER/DR5 occurs in a p53-dependent and -independent manner. However, unlike other p53-regulated genes, KILLER/DR5 is not regulated following UV irradiation. TNF-alpha, a nongenotoxic cytokine, also induced the expression of KILLER/DR5 in a number of cancer cell lines, irrespective of p53 status. TNF-alpha did not alter the KILLER/DR5 mRNA stability, suggesting that the TNF-alpha regulation of KILLER/DRS expression appears transcriptional. We also provide evidence that KILLER/DR5 is regulated in a trigger and cell type-specific manner and that its induction by TNF-alpha, p53, or DNA damage is not the consequence of apoptosis induced by these agents. Unlike KILLER/DR5, none of the other KILLER/DR5 family members, including DR4, TRID, or the ligand TRAIL, displayed genotoxic stress or TNF-alpha regulation in a p53 transcription-dependent manner. Thus, KILLER/DR5 appears a bona fide downstream target of p53 that is also regulated in a cell type-specific, trigger-dependent, and p53-independent manner.