Updated efficacy and safety of entrectinib in NTRK fusion-positive non-small cell lung cancer.

OBJECTIVES: NTRK fusions result in constitutively active oncogenic TRK proteins responsible for âˆ¼ 0.2 % of non-small cell lung cancer (NSCLC) cases. Approximately 40 % of patients with advanced NSCLC develop CNS metastases; therefore, treatments with intracranial (IC) efficacy are needed. In an integrated analysis of three phase I/II studies (ALKA-372-001: EudraCT 2012-000148-88; STARTRK-1: NCT02097810; STARTRK-2: NCT02568267), entrectinib, a potent, CNS-active, TRK inhibitor, demonstrated efficacy in patients with NTRK fusion-positive (fp) NSCLC (objective response rate [ORR]: 64.5 %; 2 August 2021 data cut-off). We present updated data for this cohort. MATERIALS AND METHODS: Eligible patients were ≥ 18 years with locally advanced/metastatic, NTRK-fp NSCLC with ≥ 12 months of follow-up. Tumor responses were assessed by blinded independent central review (BICR) per RECIST v1.1 at Week 4 and every eight weeks thereafter. Co-primary endpoints: ORR; duration of response (DoR). Secondary endpoints included progression-free survival (PFS); overall survival (OS); IC efficacy; safety. Enrolment cut-off: 2 July 2021; data cut-off: 2 August 2022. RESULTS: The efficacy-evaluable population included 51 patients with NTRK-fp NSCLC. Median age was 60.0 years (range 22-88); 20 patients (39.2 %) had investigator-assessed baseline CNS metastases. Median survival follow-up was 26.3 months (95 % CI 21.0-34.1). ORR was 62.7 % (95 % CI 48.1-75.9), with six complete and 26 partial responses. Median DoR and PFS were 27.3 months (95 % CI 19.9-30.9) and 28.0 months (95 % CI 15.7-30.4), respectively. Median OS was 41.5 months. In patients with BICR-assessed baseline CNS metastases, IC-ORR was 64.3 % (n = 9/14; 95 % CI 35.1-87.2), including seven complete responders, and IC-DoR was 55.7 months. In the safety-evaluable population (n = 55), most treatment-related adverse events were grade 1/2; no treatment-related deaths were reported. CONCLUSION: Entrectinib has continued to demonstrate deep and durable systemic and IC responses in patients with NTRK-fp NSCLC.

The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

The AML1/ETO target gene LAT2 interferes with differentiation of normal hematopoietic precursor cells.

The adaptor protein linker activator of T-cells 2 (LAT2) is a known AML1/ETO target gene whose function during normal hematopoiesis is unknown. We addressed the role of LAT2 during erythroid and myeloid differentiation of normal human CD34+ hematopoietic cells. LAT2 is expressed at low levels in CD34+ cells and upregulated during cytokine-induced myeloid and erythroid differentiation. Forced LAT2 expression leads to a delay of erythroid and myeloid differentiation keeping CD34+ cells in a more immature state, whereas LAT2 knockdown accelerates differentiation. It is tempting to speculate that by affecting the differentiation capacity of normal hematopoietic progenitors, LAT2 may contribute to the pathogenesis of AML.

MPN patients harbor recurrent truncating mutations in transcription factor NF-E2.

The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.

Transcription factor nuclear factor erythroid-2 mediates expression of the cytokine interleukin 8, a known predictor of inferior outcome in patients with myeloproliferative neoplasms.

The transcription factor nuclear factor erythroid-2 is over-expressed in patients with myeloproliferative neoplasms irrespective of the presence of the JAK2(V617F) mutation. Our transgenic mouse model over-expressing nuclear factor erythroid-2, which recapitulates many features of myeloproliferative neoplasms including transformation to acute myeloid leukemia, clearly implicates this transcription factor in the pathophysiology of myeloproliferative neoplasms. Because the targets mediating nuclear factor erythroid-2 effects are not well characterized, we conducted microarray analysis of CD34(+) cells lentivirally transduced to over-express nuclear factor erythroid-2 or to silence this transcription factor via shRNA, in order to identify novel target genes. Here, we report that the cytokine interleukin 8 is a novel target gene. Nuclear factor erythroid-2 directly binds the interleukin 8 promoter in vivo, and these binding sites are required for promoter activity. Serum levels of interleukin 8 are known to be elevated in both polycythemia vera and primary myelofibrosis patients. Recently, increased interleukin 8 levels have been shown to be predictive of inferior survival in primary myelofibrosis patients in multivariate analysis. Therefore, one of the mechanisms by which nuclear factor erythroid-2 contributes to myeloproliferative neoplasm pathology may be increased interleukin 8 expression.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. 'BH3-only' proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

p38ß-regulated induction of the heat shock response by carbon monoxide releasing molecule CORM-2 mediates cytoprotection in lung cells in vitro.

The carbon monoxide releasing molecule tricarbonyldichlororuthenium (CORM-2) displays protective actions like carbon monoxide. The molecular mechanism underlying this effect remains controversial. We hypothesized that CORM-2 mediates cytoprotection via induction of heat shock proteins through activation of p38 mitogen-activated kinase. Embryonic bovine lung cells were incubated with CORM-2. Apoptosis was induced by staurosporine and analyzed by flow cytometry following annexin-V staining, caspase-3 activity assay, and by Western Blot for caspase-3 cleavage. Heat shock response was assessed by DNA-binding activity of heat shock factor 1 and by reporter gene activity. Cells were transfected with siRNA targeting p38 isoforms. Data were analyzed with ANOVA and post-hoc Holm-Sidak test. CORM-2 inhibited staurosporine-induced apoptosis (% annexin-V positive cells: staurosporine = 60 ± 4% vs. CORM-2 10 µM = 48 ± 4%, CORM-2 25 µM=42 ± 5%, CORM-2 50 µM = 40 ± 4% and CORM-2 100 µM = 38 ± 2%, mean ± S.D., P<0.001; caspase-3 activity: staurosporine=92 ± 15 RFUs vs. CORM-2 50 µM=60 ± 14 RFUs, mean ± S.D. P<0.001). CORM-2 induced phosphorylation of p38 MAPK, but not of JNK and ERK1/2. CORM-2 induced DNA-binding of heat shock factor 1 and elicited a 4-fold induction of gene activity (P<0.05). Incubation with the Hsp inhibitors KNK437 attenuated and 17-AAG abolished the anti-apoptotic effect of CORM-2 (P<0.001). p38 inhibition and silencing of p38ß attenuated the anti-apoptotic effect of CORM-2 (P<0.05), most likely by abolishing CORM-2-induced HSF-1 binding activity. These findings suggest that CORM-2-mediated cytoprotection is caused by induction of the heat shock response and by p38 activation. Furthermore, the p38ß isoform activation may represent an upstream mechanism of heat shock response induction.

AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2.

The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2(V617F) mutation. Although NF-E2 levels correlate with JAK2(V671F) allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2(V617F) mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2(V617F). In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-beta significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients.

JAK2V617F-negative ET patients do not display constitutively active JAK/STAT signaling.

OBJECTIVE: Presence of the JAK2V617F mutation in only 40% to 60% of patients with essential thrombocythemia (ET) underscores the heterogeneity of this myeloproliferative disorder (MPD). Several distinct mutations, either in JAK2 (exon 12) or in c-Mpl (W515L) have been described in subsets of other MPDs, polycythemia vera, and idiopathic myelofibrosis. Analogous to JAK2,V617F these mutations cause constitutive JAK2 and signal transducer and activation of transcription (STAT) activation. It has therefore been proposed that constitutive activation of the JAK/STAT pathway underlies the molecular etiology of all MPDs. We investigated the alternative hypothesis that distinct alterations, separate from the JAK/STAT signal transduction pathway, underlie a subset of JAK2V617F-negative ET. METHODS: cDNA microarrays and quantitative reverse transcriptase polymerase chain reactions were used to compare gene expression in 40 ET patients with and without the JAK2V617F mutation. RESULTS: Unsupervised clustering of gene-expression patterns in ET patients revealed two distinct subclasses of patients. These subclasses differed in presence or absence of the JAK2V617F mutation. Patients lacking the JAK2V617F mutation displayed significantly lower expression of the JAK/STAT target genes Pim-1 and suppressor of cytokine signaling-2. In addition, JAK2V617F-negative patients showed lower levels of STAT3 phosphorylation. CONCLUSIONS: These data demonstrate that a large proportion of JAK2V617F-negative ET patients do not display constitutive JAK/STAT signaling. Hence, we propose that alterations in different signal transduction pathways can lead to the clinical phenotype of ET. Elucidation of novel ET-inducing changes will facilitate both a molecular classification of ET and development of rationally designed therapies.