Pediatr Blood Cancer
Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
BACKGROUND: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. METHODS: One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. RESULTS: Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. CONCLUSION: This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.
Nature reviews. Cancer
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Nat Rev Clin Oncol
ATP-competitive Fibroblast Growth Factor Receptor (FGFR) kinase inhibitors, including BGJ398 and Debio1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor, TAS-120, demonstrated efficacy in four patients with FGFR2-fusion-positive ICC who developed resistance to BGJ398 or Debio1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsies and ctDNA, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC.
Identifying drug resistance mutations is important for the clinical use of antivirals and can help define both a drug's mechanism of action and the mechanistic basis of resistance. Resistance mutations are often identified one-at-a-time by studying viral evolution within treated patients or during viral growth in the presence of a drug in cell culture. Such approaches have previously mapped resistance to enfuvirtide, the only clinically approved HIV-1 fusion inhibitor, to enfuvirtide's binding site in the N-terminal heptad repeat (NHR) of the Envelope (Env) transmembrane domain as well as a limited number of allosteric sites. Here, we sought to better delineate the genotypic determinants of resistance throughout Env. We used deep mutational scanning to quantify the effect of all single-amino-acid mutations to the subtype A BG505 Env on resistance to enfuvirtide. We identified both previously characterized and numerous novel resistance mutations in the NHR. Additional resistance mutations clustered in other regions of Env conformational intermediates, suggesting they may act during different fusion steps by altering fusion kinetics and/or exposure of the enfuvirtide binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and highlights the utility of using deep mutational scanning to comprehensively map potential drug resistance mutations.
Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer (PCa) initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary PCa. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in PCa relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice-variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small molecule SR-BI antagonist Block Lipid Transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and non-steroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC.
The Journal of biological chemistry
Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates alpha1-adrenergic receptor (alpha1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for alpha1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr(198) We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr(198)-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr(198) by SRC. We specifically detected SRC-mediated Tyr(198) phosphorylation at the plasma membrane and observed that it is not enhanced or induced by alpha1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr(198) phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based alpha1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr(198)-phosphorylated PANX1 is involved in hypertensive vascular pathology.
Castration-resistant prostate cancer (CRPC) that has developed resistance to the new-generation androgen receptor (AR) antagonist enzalutamide is a lethal disease. Transcriptome analysis of multiple prostate cancer models identified CXCR7, an atypical chemokine receptor, as one of the most upregulated genes in enzalutamide-resistant cells. AR directly repressed CXCR7 by binding to an enhancer 110kb downstream of the gene and expression was restored upon androgen deprivation. We demonstrate that CXCR7 is a critical regulator of prostate cancer sensitivity to enzalutamide and is required for CRPC growth in vitro and in vivo. Elevated CXCR7 activated MAPK/ERK signaling through ligand-independent but beta-arrestin 2-dependent mechanisms. Examination of patient specimens showed that CXCR7 and pERK levels increased significantly from localized prostate cancer to CRPC and further upon enzalutamide resistance. Preclinical studies revealed remarkable efficacies of MAPK/ERK inhibitors in suppressing enzalutamide-resistant prostate cancer. Overall, these results indicate that CXCR7 may serve as a biomarker of resistant disease in patients with prostate cancer and that disruption of CXCR7 signaling may be an effective strategy to overcome resistance.
Nature reviews. Cancer
Small cell lung cancer (SCLC) is an exceptionally lethal malignancy for which more effective therapies are urgently needed. Several lines of evidence, from SCLC primary human tumours, patient-derived xenografts, cancer cell lines and genetically engineered mouse models, appear to be converging on a new model of SCLC subtypes defined by differential expression of four key transcription regulators: achaete-scute homologue 1 (ASCL1; also known as ASH1), neurogenic differentiation factor 1 (NeuroD1), yes-associated protein 1 (YAP1) and POU class 2 homeobox 3 (POU2F3). In this Perspectives article, we review and synthesize these recent lines of evidence and propose a working nomenclature for SCLC subtypes defined by relative expression of these four factors. Defining the unique therapeutic vulnerabilities of these subtypes of SCLC should help to focus and accelerate therapeutic research, leading to rationally targeted approaches that may ultimately improve clinical outcomes for patients with this disease.