Safgren, Stephanie L

ABSTRACT The cohesin complex, composed of SMC1, SMC3, RAD21, and STAG1/STAG2, is essential for chromosome cohesion, DNA repair, and transcriptional regulation. Pathogenic variants in cohesin components cause cohesinopathies. The classical characteristics of cohesinopathies include developmental delay (DD), intellectual disability (ID), feeding difficulties, hypotonia, short stature, hearing loss, and dysmorphic features. Here, we present a 5‐year‐old boy with classical cohesinopathy features, including DD/ID and feeding difficulties, along with non‐classical features such as hypoglycemia, recurrent fever, and immunodeficiency. Trio exome sequencing identified a novel de novo missense variant of uncertain significance (NM_005862.3:c.643G>A(p.Val215Ile)) in the STAG1 gene. The variant localizes to the RAD21 interaction interface, and molecular dynamics (MD) simulations revealed conformational changes comparable to other STAG1 variants reported as likely pathogenic in patients, supporting a deleterious effect which may disrupt the STAG1‐RAD21 interaction interface. This case expands the phenotypic and molecular spectrum of STAG1‐related cohesinopathy and advances our understanding of the disease mechanism.

PURPOSE Human epidermal growth factor receptor 2 (HER2), a transmembrane receptor with kinase activity involved in cell signaling through the RAS pathway, is expressed in osteosarcoma (OST). Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate with a HER2-targeting monoclonal antibody, trastuzumab, linked to a topoisomerase I inhibitor. We report results of a phase II study of T-DXd in adolescents and young adults with HER2-positive relapsed, unresectable OST (ClinicalTrials.gov identifier: NCT04616560 ). METHODS Patients age 12-39 years with relapsed, unresectable OST were eligible for centrally evaluated HER2 expression screening by immunohistochemistry. Patients with >10% of OST cells with cytoplasmic or membranous HER2 expression of any intensity were eligible to receive T-DXd at 5.4 mg/kg intravenously once every 3 weeks for up to 2 years. The study used a 9 + 15 Simon's optimal two-stage design with a primary end point of event-free survival at 24 weeks. RESULTS Fifty patients were screened for HER2, and 41 met the inclusion criteria of 10% of OST cells having membranous or cytoplasmic HER2 expression. Nine eligible patients, two younger than 18 years, were enrolled and received T-DXd. With a single patient having more than 24 weeks of stable disease (SD) of the nine planned in the first stage, this study did not meet criteria to progress to stage II. The estimated SD rate at 24 weeks is 11.1%. The median (range) number of cycles of therapy was 2 (2-12). Grade 3 and 4 possibly related adverse events occurred in three of nine patients and included nausea, vomiting, tumor hemorrhage, cytopenias (n = 3), wound infection, and hypertension. CONCLUSION T-DXd did not demonstrate sufficient activity to expand enrollment to the planned second stage in patients with OST. No new safety signals were observed.

Abstract Purpose: Selinexor is a first-in-class, central nervous system (CNS)–penetrant, oral inhibitor of exportin 1 (XPO1), the main nuclear exporter of many key tumor suppressors. We report a phase I trial of selinexor in children and adolescents with recurrent CNS and solid tumors (NCT02323880). Patients and Methods: A rolling six design was used to evaluate the maximum tolerated dose (MTD) and first dose pharmacokinetics of selinexor administered once (35–45 mg/m2) or twice (20–35 mg/m2) weekly during a 28-day cycle (part A). Ten additional patients with high-grade glioma (HGG) were treated at the MTD administered once weekly (part B). Results: In part A, 49 patients were enrolled. Continuous twice weekly dosing was limited by extended hematologic toxicity. The MTD on a twice weekly schedule for 3 weeks on/1 week off (twice weekly 3/1) was 20 mg/m2/dose. Dose-limiting toxicities (DLTs) on this schedule included fatigue, acute reversible neurologic changes, neutropenia, thrombocytopenia, and aspartate aminotransferase/alanine aminotransferase increase. On a continuous once weekly schedule, the MTD was 35 mg/m2/dose; DLTs included seizure and thrombocytopenia. In part B (HGG expansion), there were no additional DLTs observed. Non-DLTs included lymphopenia, leukopenia, neutropenia, thrombocytopenia, anorexia, fatigue, hypophosphatemia, nausea, and vomiting. There were no objective responses. The median number of cycles received was 1 (range, 1–9); eight of 59 patients (13.5%) received 5 to 9 cycles, five of whom had HGG. Conclusions: Selinexor-related toxicities were primarily hematologic and neurologic, requiring dose or dose-frequency reduction. The MTD and recommended initial phase II dose of selinexor in children and adolescents with recurrent solid and CNS tumors is 35 mg/m2/dose once weekly.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell–specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

AbstractAn infant was referred for evaluation of congenital glaucoma and corneal clouding. In addition, he had a pelvic kidney, hypotonia, patent ductus arteriosus, abnormal pinnae, and developmental delay. Exome sequencing identified a previously unpublished de novo single nucleotide insertion in PBX1 c.400dupG (NM_002585.3), predicted to cause a frameshift resulting in a truncated protein with loss of function (p.Ala134Glyfs*65). Identification of this loss of function variant supports the diagnosis of congenital anomalies of the kidney and urinary tract syndrome with or without hearing loss, abnormal ears, or developmental delay (CAKUTHED). Here, we propose glaucoma as an extra‐renal manifestation associated with PBX1‐related disease due to the relationship of PBX1 with MEIS1, MEIS2, and FOXC1 transcription factors associated with eye development.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Abstract Purpose: Aurora A kinase (AAK) plays an integral role in mitotic entry, DNA damage checkpoint recovery, and centrosome and spindle maturation. Alisertib (MLN8237) is a potent and selective AAK inhibitor. In pediatric preclinical models, antitumor activity was observed in neuroblastoma, acute lymphoblastic leukemia, and sarcoma xenografts. We conducted a phase 2 trial of alisertib in pediatric patients with refractory or recurrent solid tumors or acute leukemias (NCT01154816). Patients and Methods: Alisertib (80 mg/m2/dose) was administered orally, daily for 7 days every 21 days. Pharmacogenomic (PG) evaluation for polymorphisms in the AURK gene and drug metabolizing enzymes (UGT1A1*28), and plasma pharmacokinetic studies (PK) were performed. Using a 2-stage design, patients were enrolled to 12 disease strata (10 solid tumor and 2 acute leukemia). Response was assessed after cycle 1, then every other cycle. Results: A total of 139 children and adolescents (median age, 10 years) were enrolled, 137 were evaluable for response. Five objective responses were observed (2 complete responses and 3 partial responses). The most frequent toxicity was myelosuppression. The median alisertib trough concentration on day 4 was 1.3 μmol/L, exceeding the 1 μmol/L target trough concentration in 67% of patients. No correlations between PG or PK and toxicity were observed. Conclusions: Despite alisertib activity in pediatric xenograft models and cogent pharmacokinetic-pharmacodynamic relationships in preclinical models and adults, the objective response rate in children and adolescents receiving single-agent alisertib was less than 5%.

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor–positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.

Abstract Purpose: Controversy exists about CYP2D6 genotype and tamoxifen efficacy. Experimental Design: A matched case–control study was conducted using the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG8) that randomized postmenopausal women with estrogen receptor (ER)-positive breast cancer to tamoxifen for 5 years (arm A) or tamoxifen for 2 years followed by anastrozole for 3 years (arm B). Cases had disease recurrence, contralateral breast cancer, second non–breast cancer, or died. For each case, controls were identified from the same treatment arm of similar age, surgery/radiation, and tumor–node—metastasis (TNM) stage. Genotyping was conducted for alleles associated with no (PM; *3, *4, *6), reduced (IM; *10, and *41), and extensive (EM: absence of these alleles) CYP2D6 metabolism. Results: The common CYP2D6*4 allele was in Hardy–Weinberg equilibrium. In arm A during the first 5 years of therapy, women with two poor alleles [PM/PM: OR, 2.45; 95% confidence interval (CI), 1.05–5.73, P = 0.04] and women with one poor allele (PM/IM or PM/EM: OR, 1.67; 95% CI, 0.95–2.93; P = 0.07) had a higher likelihood of an event than women with two extensive alleles (EM/EM). In years 3 to 5 when patients remained on tamoxifen (arm A) or switched to anastrozole (arm B), PM/PM tended toward a higher likelihood of a disease event relative to EM/EM (OR, 2.40; 95% CI, 0.86–6.66; P = 0.09) among women on arm A but not among women on arm B (OR, 0.28; 95% CI, 0.03–2.30). Conclusion: In ABCSG8, the negative effects of reduced CYP2D6 metabolism were observed only during the period of tamoxifen administration and not after switching to anastrozole. Clin Cancer Res; 19(2); 500–7. ©2012 AACR.