Patterns of Ciliation and Ciliary Signaling in Cancer.

Among the factors that have been strongly implicated in regulating cancerous transformation, the primary monocilium (cilium) has gained increasing attention. The cilium is a small organelle extending from the plasma membrane, which provides a localized hub for concentration of transmembrane receptors. These receptors transmit signals from soluble factors (including Sonic hedgehog (SHH), platelet-derived growth factor (PDGF-AA), WNT, TGFß, NOTCH, and others) that regulate cell growth, as well as mechanosensory cues provided by flow or extracellular matrix. Ciliation is regulated by cell cycle, with most cells that are in G0 (quiescent) or early G1 ciliation and cilia typically absent in G2/M cells. Notably, while most cells organized in solid tissues are ciliated, cancerous transformation induces significant changes in ciliation. Most cancer cells lose cilia; medulloblastomas and basal cell carcinomas, dependent on an active SHH pathway, rely on ciliary maintenance. Changes in cancer cell ciliation are driven by core oncogenic pathways (EGFR, KRAS, AURKA, PI3K), and importantly ciliation status regulates functionality of those pathways. Ciliation is both influenced by targeted cancer therapies and linked to therapeutic resistance; recent studies suggest ciliation may also influence cancer cell metabolism and stem cell identity. We review recent studies defining the relationship between cilia and cancer.

Combined inhibition of Aurora A and p21-activated kinase 1 as a new treatment strategy in breast cancer.

PURPOSE: The serine-threonine kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast tumors, with overexpression promoting aggressive breast cancer phenotypes and poor clinical outcomes. Besides the well-defined roles of these proteins in control of cell division, proliferation, and invasion, both kinases support MAPK kinase pathway activation and can contribute to endocrine resistance by phosphorylating estrogen receptor alpha (ERα). PAK1 directly phosphorylates AURKA and its functional partners, suggesting potential value of inhibiting both kinases activity in tumors overexpressing PAK1 and/or AURKA. Here, for the first time, we evaluated the effect of combining the AURKA inhibitor alisertib and the PAK inhibitor FRAX1036 in preclinical models of breast cancer. METHODS: Combination of alisertib and FRAX1036 was evaluated in a panel of 13 human breast tumor cell lines and BT474 xenograft model, with assessment of the cell cycle by FACS, and signaling changes by immunohistochemistry and Western blot. Additionally, we performed in silico analysis to identify markers of response to alisertib and FRAX1036. RESULTS: Pharmacological inhibition of AURKA and PAK1 synergistically decreased survival of multiple tumor cell lines, showing particular effectiveness in luminal and HER2-enriched models, and inhibited growth and ERα-driven signaling in a BT474 xenograft model. In silico analysis suggested cell lines with dependence on AURKA are most likely to be sensitive to PAK1 inhibition. CONCLUSION: Dual targeting of AURKA and PAK1 may be a promising therapeutic strategy for treatment of breast cancer, with a particular effectiveness in luminal and HER2-enriched tumor subtypes.

Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-d-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression.-Nikonova, A. S., Deneka, A. Y., Kiseleva, A. A., Korobeynikov, V., Gaponova, A., Serebriiskii, I. G., Kopp, M. C., Hensley, H. H., Seeger-Nukpezah, T. N., Somlo, S., Proia, D. A., Golemis, E. A. Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Musashi-2 (MSI2) supports TGF-ß signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-ß receptor 1 (TGFßR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFßRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFßR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Nedd9 restrains renal cystogenesis in Pkd1-/- mice.

Mutations inactivating the cilia-localized Pkd1 protein result in autosomal dominant polycystic kidney disease (ADPKD), a serious inherited syndrome affecting ∼ 1 in 500 people, in which accumulation of renal cysts eventually destroys kidney function. Severity of ADPKD varies throughout the population, for reasons thought to involve differences both in intragenic Pkd1 mutations and in modifier alleles. The scaffolding protein NEDD9, commonly dysregulated during cancer progression, interacts with Aurora-A (AURKA) kinase to control ciliary resorption, and with Src and other partners to influence proliferative signaling pathways often activated in ADPKD. We here demonstrate Nedd9 expression is deregulated in human ADPKD and a mouse ADPKD model. Although genetic ablation of Nedd9 does not independently influence cystogenesis, constitutive absence of Nedd9 strongly promotes cyst formation in the tamoxifen-inducible Pkd1fl/fl;Cre/Esr1(+) mouse model of ADPKD. This cystogenic effect is associated with striking morphological defects in the cilia of Pkd1(-/-);Nedd9(-/-) mice, associated with specific loss of ciliary localization of adenylase cyclase III in the doubly mutant genotype. Ciliary phenotypes imply a failure of Aurora-A activation: Compatible with this idea, Pkd1(-/-);Nedd9(-/-) mice had ciliary resorption defects, and treatment of Pkd1(-/-) mice with a clinical Aurora-A kinase inhibitor exacerbated cystogenesis. In addition, activation of the ADPKD-associated signaling effectors Src, Erk, and the mTOR effector S6 was enhanced, and Ca(2+) response to external stimuli was reduced, in Pkd1(-/-);Nedd9(-/-) versus Pkd1(-/-) mice. Together, these results indicated an important modifier action of Nedd9 on ADPKD pathogenesis involving failure to activate Aurora-A.

A New Sensitive and Selective Off-On Fluorescent Zn2+ Chemosensor Based on 3,3',5,5'-Tetraphenylsubstituted Dipyrromethene.

3,3',5,5'-Tetraphenyl-2,2'-dipyrromethene was described as a highly sensitive and selective Off-on fluorescent colorimetric chemosensor for Zn2+ based on the chelation-enhanced fluorescence (CHEF) effect. The reaction of dipyrromethene ligand with Zn2+ induces the formation of the [ZnL2] complex, which exhibits the increasing fluorescence in 120 fold compared with ligand in the propanol-1/cyclohexane (1:30) binary mixture. The Zn2+ detection limit was 1.4 × 10-7 М. The UV-Vis and fluorescence spectroscopic studies demonstrated that the dipyrromethene sensor was highly selective toward Zn2+ cations over other metal ions (Na+, Mg2+, Co2+, Ni2+, Fe3+, Cu2+, Mn2+, Cd2+ and Pb2+), excluding Hg2+.

Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a progressive genetic syndrome with an incidence of 1:500 in the population, arising from inherited mutations in the genes for polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2). Typical onset is in middle age, with gradual replacement of renal tissue with thousands of fluid-filled cysts, resulting in end-stage renal disease requiring dialysis or kidney transplantation. There currently are no approved therapies to slow or cure ADPKD. Mutations in the PKD1 and PKD2 genes abnormally activate multiple signaling proteins and pathways regulating cell proliferation, many of which we observe, through network construction, to be regulated by heat shock protein 90 (HSP90). Inhibiting HSP90 with a small molecule, STA-2842, induces the degradation of many ADPKD-relevant HSP90 client proteins in Pkd1(-/-) primary kidney cells and in vivo. Using a conditional Cre-mediated mouse model to inactivate Pkd1 in vivo, we find that weekly administration of STA-2842 over 10 wk significantly reduces initial formation of renal cysts and kidney growth and slows the progression of these phenotypes in mice with preexisting cysts. These improved disease phenotypes are accompanied by improved indicators of kidney function and reduced expression and activity of HSP90 clients and their effectors, with the degree of inhibition correlating with cystic expansion in individual animals. Pharmacokinetic analysis indicates that HSP90 is overexpressed and HSP90 inhibitors are selectively retained in cystic versus normal kidney tissue, analogous to the situation observed in solid tumors. These results provide an initial justification for evaluating HSP90 inhibitors as therapeutic agents for ADPKD.

Predictors of delay in diagnosis and treatment in diffuse large B-cell lymphoma and impact on survival.

There is a paucity of data on the impact of diagnostic and treatment delays on outcomes in haematological malignancies, particularly in patients with diffuse large B-cell lymphoma (DLBCL). Our database of patients treated for DLBCL between 2002 and 2010 was interrogated. Univariate and multivariate analyses were performed to determine the relationship between sociodemographic or disease-specific variables and delays. Cox Regression analysis was used to discern the impact of delays on survival. Patients (n = 278) waited a median of 4 weeks before seeking medical attention. It took a median of 8 weeks for a non-haematology physician to diagnose DLBCL and refer to a haematologist. A median of 3 weeks elapsed between specialist consultation and chemotherapy initiation. In multivariate logistic regression analysis, bone marrow involvement [odds ratio (OR) = 0·41, P = 0·018], Charlson comorbidity index (OR = 1·42, P = 0·017) and urgent inpatient chemotherapy (OR = 0·40, P = 0·012) were associated with diagnostic delays >6 weeks. Lack of pathological diagnosis at the time of haematology referral was the only factor that independently predicted for treatment delays >4 weeks (OR = 8·25, P < 0·01). Diagnostic or treatment delays did not impact survival or progression-free survival. In conclusion, selected disease and patient-related factors are associated with delays in management of DLBCL, but do not impact outcomes.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

BACKGROUND: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. METHODS: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. RESULTS: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. CONCLUSIONS: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

CAS proteins in health and disease: an update.

The CAS family of scaffolding proteins has increasingly attracted scrutiny as important for regulation of cancer-associated signaling. BCAR1 (also known as p130Cas), NEDD9 (HEF1, Cas-L), EFS (Sin), and CASS4 (HEPL) are regulated by and mediate cell attachment, growth factor, and chemokine signaling. Altered expression and activity of CAS proteins are now known to promote metastasis and drug resistance in cancer, influence normal development, and contribute to the pathogenesis of heart and pulmonary disease. In this article, we provide an update on recently published studies describing signals regulating and regulated by CAS proteins, and evidence for biological activity of CAS proteins in normal development, cancer, and other pathological conditions.

Aurora A kinase (AURKA) in normal and pathological cell division.

Temporally and spatially controlled activation of the Aurora A kinase (AURKA) regulates centrosome maturation, entry into mitosis, formation and function of the bipolar spindle, and cytokinesis. Genetic amplification and mRNA and protein overexpression of Aurora A are common in many types of solid tumor, and associated with aneuploidy, supernumerary centrosomes, defective mitotic spindles, and resistance to apoptosis. These properties have led Aurora A to be considered a high-value target for development of cancer therapeutics, with multiple agents currently in early-phase clinical trials. More recently, identification of additional, non-mitotic functions and means of activation of Aurora A during interphase neurite elongation and ciliary resorption have significantly expanded our understanding of its function, and may offer insights into the clinical performance of Aurora A inhibitors. Here we review the mitotic and non-mitotic functions of Aurora A, discuss Aurora A regulation in the context of protein structural information, and evaluate progress in understanding and inhibiting Aurora A in cancer.

Management of Marginal Zone Lymphoma: A Canadian Perspective.

Marginal zone lymphomas (MZL) are a rare, heterogenous group of lymphomas, accounting for 5-17% of indolent non-Hodgkin lymphomas in the western world. They can be further divided into three subtypes: extranodal MZL, splenic MZL, and nodal MZL. These subtypes differ in clinical presentation and behavior, which influences how they are managed. There is currently no standard of care for the treatment of MZL, owing to the difficulty in conducting phase 3 randomized trials in MZL, and the fact that there are limited data on the efficacy of therapy in individual subtypes. Treatment practices are thus largely borrowed from other indolent lymphomas and are based on patient and disease characteristics, as well as access to therapy. This review summarizes the Canadian treatment landscape for MZL and how these therapies may be sequenced in practice.

Loss of Pkd1 limits susceptibility to colitis and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~50,000 deaths. Autosomal dominant polycystic kidney disease (ADPKD), associated with mutations disabling the PKD1 gene, affects as many as 1 in 1000. Intriguingly, some studies have suggested that individuals with germline mutations in PKD1 have reduced incidence of CRC, suggesting a genetic modifier function. Using mouse models, we here establish that loss of Pkd1 greatly reduces CRC incidence and tumor growth induced by loss of the tumor suppressor Apc. Growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/- organoids, indicating a cancer cell-intrinsic activity, even though Pkd1 loss enhanced activity of pro-oncogenic signaling pathways. Notably, Pkd1 loss increased colon barrier function, with Pkd1-deficient animals resistant to DSS-induced colitis, associated with upregulation of claudins that decrease permeability, and reduced T cell infiltration. Notably, Pkd1 loss caused greater sensitivity to activation of CFTR, a tumor suppressor in CRC, paralleling signaling relations in ADPKD. Overall, these data and other data suggest germline and somatic mutations in PKD1 may influence incidence, presentation, and treatment response in human CRC and other pathologies involving the colon.

Gaining Wings to FLY: Using Drosophila Oogenesis as an Entry Point for Citizen Scientists in Laboratory Research.

Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.

NEDD9 sustains hexokinase expression to promote glycolysis.

Elevated rates of glycolysis in cancer cells support tumor growth, in a process that typically depends on oncogene-induced increases in the expression and/or activity of enzymes in the glycolytic pathway. The NEDD9 scaffolding protein is upregulated in many advanced tumors, with increased NEDD9 promoting the activity of SRC and other effectors that promote invasion and metastasis. We here define a new role for NEDD9 in support of glycolysis. NEDD9 knockdown significantly impaired glycolysis in multiple lung cancer cell lines This was accompanied by post-transcriptional downregulation of steady-state levels of hexokinases (HK1 and HK2), which catalyze early steps in the glycolytic cascade, key rate limiting enzyme phosphofructokinase (PFK1), and downstream glyceraldehyde phosphate dehydrogenase (GAPDH). In mice, protein levels of HK1, HK2, PFK1, and GAPDH were depressed in Krastm4Tyj/J /Trp53tm1Brn/J (KP) non-small cell lung tumors with null versus wild type Nedd9. Reciprocally, depletion of HK1 or HK2 elevated NEDD9 expression, as did the treatment of cells with 2-deoxyglucose (2DG), an inhibitor of glycolysis; whereas overexpression of hexokinases promoted NEDD9 dephosphorylation, associated with reduced NEDD9 activity. Together, these data for the first time suggest a negative feedback circuit involving NEDD9 and glycolytic enzymes that may contribute to NEDD9 action in promoting the aggressive growth of advanced tumors.

Aldosterone-induced activation of signaling pathways requires activity of angiotensin type 1a receptors.

RATIONALE: Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)-mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown. OBJECTIVES: We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) would decrease cellular effects induced by aldosterone. METHODS AND RESULTS: We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT(1a)R knockout mice. CONCLUSION: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Evaluation of the Small-molecule BRD4 Degrader CFT-2718 in Small-cell Lung Cancer and Pancreatic Cancer Models.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.