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Simple Summary Tumor cells communicate with the stromal cells within the tumor microenvironment (TME) to create a conducive environment for tumor growth. One major avenue for mediating crosstalk between various cell types in the TME involves exchanges of molecular payloads in the form of extracellular vesicles/exosomes. Autophagy is a fundamental mechanism to maintain intracellular homeostasis but recent reports suggest that secretory autophagy plays an important role in promoting secretion of exosomes that are packaged with non-coding RNAs (ncRNAs) and other biomolecules from the donor cell. Uptake of exosomal autophagy-modulating ncRNAs by recipient cells may further perpetuate tumor progression. Abstract Autophagy is a fundamental cellular homeostasis mechanism known to play multifaceted roles in the natural history of cancers over time. It has recently been shown that autophagy also mediates the crosstalk between the tumor and its microenvironment by promoting the export of molecular payloads such as non-coding RNA (ncRNAs) via LC3-dependent Extracellular Vesicle loading and secretion (LDELS). In turn, the dynamic exchange of exosomal ncRNAs regulate autophagic responses in the recipient cells within the tumor microenvironment (TME), for both tumor and stromal cells. Autophagy-dependent phenotypic changes in the recipient cells further enhance tumor growth and metastasis, through diverse biological processes, including nutrient supplementation, immune evasion, angiogenesis, and therapeutic resistance. In this review, we discuss how the feedforward autophagy-ncRNA axis orchestrates vital communications between various cell types within the TME ecosystem to promote cancer progression.

Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5′-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5′-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5′-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5′-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.

Transfer RNA (tRNA)‐derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell‐state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation‐dependent enrichment of 5′‐tsRNAs. We report the identification of a set of 5′‐tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5′‐tsRNAs revealed a switch in their association with “effector” RNPs and “target” mRNAs in different cell states. We demonstrate that specific 5′‐tsRNAs can preferentially interact with the RNA‐binding protein, Igf2bp1, in the RA‐induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency‐promoting factor, c‐Myc, thus providing a mechanistic basis for how 5′‐tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5′‐tsRNAs in defining distinct cell states.

MicroRNAs (miRNAs) are short single-stranded RNAs that have pivotal roles in disease pathophysiology through transcriptional and translational modulation of important genes. It has been implicated in the development of many diseases, such as stroke, cardiovascular conditions, cancers and inflammatory airway diseases. There is recent evidence that miRNAs play important roles in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD), and could help to distinguish between T2-low (non-eosinophilic, steroid-insensitive) versus T2-high (eosinophilic, steroid-sensitive) disease endotypes. As these are the two most prevalent chronic respiratory diseases globally, with rising disease burden, miRNA research might lead to the development of new diagnostic and therapeutic targets. Research involving miRNAs in airway disease is challenging because: (i) asthma and COPD are heterogeneous inflammatory airway diseases; there are overlapping but distinct inter- and intra-disease differences in the immunological pathophysiology, (ii) there exists more than 2,000 known miRNAs and a single miRNA can regulate multiple targets, (iii) differential effects of miRNAs could be present in different cellular subtypes and tissues, and (iv) dysregulated miRNA expression might be a direct consequence of an indirect effect of airway disease onset or progression. As miRNAs are actively secreted in fluids and remain relatively stable, they have the potential for biomarker development and therapeutic targets. In this review, we summarize the preclinical data on potential miRNA biomarkers that mediate different pathophysiological mechanisms in airway disease. We discuss the framework for biomarker development using miRNA and highlight the need for careful patient characterization and endotyping in the screening and validation cohorts, profiling both airway and blood samples to determine the biological fluids of choice in different disease states or severity, and adopting an untargeted approach. Collaboration between the various stakeholders - pharmaceutical companies, laboratory professionals and clinician-scientists is crucial to reduce the difficulties and cost required to bring miRNA research into the translational stage for airway diseases.

The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients.

Despite recent advances in breast cancer therapeutics, mortality of metastatic triple negative breast cancer (TNBC) subtype remains high; due to their lack of hormone receptors expression for targeted therapy. Aberrant activation of Wnt/β-catenin signaling has been associated with breast cancers; where 40% of total breast cancers have elevated β-catenin levels with increased Wnt activity. Recently, we identified DEAD-box RNA helicase DP103 as a novel prognostic biomarker and metastasis-driving oncogene; highly expressed in TNBC subtype. Interestingly, we found high DP103 expression to be positively correlated with high β-catenin expression in clinical specimens (n=400). This led us to hypothesize a possible role of DP103 in modulating the Wnt/β-catenin pathway in TNBCs. Depletion of DP103 in metastatic TNBC cells decreases Wnt/β-catenin activity and expression of downstream Wnt target genes, while overexpression of DP103 increases Wnt activity. Depletion of DP103 also decreases phosphorylation of LRP6 and several important Wnt modulators required for downstream Wnt activation. Moreover, induction of Wnt/β-catenin signaling in Wnt responsive TNBC cells also significantly increased DP103 expression, indicating a possible positive feedback loop. Both canonical and non-canonical Wnt signaling is known to independently promote stem cell growth in mammospheres. Herein, we will also provide evidence on the role of DP103 in promoting breast cancer stem cell-like properties. Collectively, our data show a novel regulatory role of DP103 in the Wnt/β-catenin signaling pathway and in promoting breast cancer stem cell-like behavior, presenting itself as a potential drug target in TNBC patients. Citation Format: Cai W, Cheong JK, Edison E, Banerjee A, Tan TZ, Gaboury L, Yousef EM, Thiery JP, Lobie PE, Virshup DM, Yap CT, Kumar AP. DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-03.

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Here we report the identification of a set of tsRNAs upregulated in differentiating mouse embryonic stem cells (mESCs). Mechanistic analyses revealed primary functions of tsRNAs in regulating polysome assembly and translation. Notably, interactome studies with differentially-enriched tsRNAs revealed a switch in associations with ‘effector’ RNPs and ‘target’ mRNAs in different cell-states. We also demonstrate that a specific pool of tsRNAs can interact with Igf2bp1, an RNA-binding protein, to influence the expression of the pluripotency-promoting factor-c-Myc, thereby providing evidence for tsRNAs in modulating stem cell-states in mESCs. Finally, tsRNA expression analyses in distinct, heterologous cell and tissue models of stem/transformed versus differentiated/normal states reveal that tsRNA-mediated regulation of protein translation may represent a global biological phenomenon associated with cell-state transitions.One Sentence SummaryIdentification and functional characterization of tRNA-derived small RNAs (tsRNAs) in cell state switches.

Evasion of forkhead box O (FOXO) family of longevity-related transcription factors-mediated growth suppression is necessary to promote cancer development. Since somatic alterations or mutations and transcriptional dysregulation of the FOXO genes are infrequent in human cancers, it remains unclear how these tumour suppressors are eliminated from cancer cells. The protein stability of FOXO3A is regulated by Casein Kinase 1 alpha (CK1α) in an oncogenic RAS-specific manner, but whether this mode of regulation extends to related FOXO family members is unknown. Here we report that CK1α similarly destabilizes FOXO4 in RAS-mutant cells by phosphorylation at serines 265/268. The CK1α-dependent phosphoregulation of FOXO4 is primed, in part, by the PI3K/AKT effector axis of oncogenic RAS signalling. In addition, mutant RAS coordinately elevates proteasome subunit expression and proteolytic activity to eradicate nuclear FOXO4 proteins from RAS-mutant cancer cells. Importantly, dual inhibition of CK1α and the proteasome synergistically inhibited the growth of multiple RAS-mutant human cancer cell lines of diverse tissue origin by blockade of nuclear FOXO4 degradation and induction of caspase-dependent apoptosis. Our findings challenge the current paradigm that nuclear export regulates the proteolysis of FOXO3A/4 tumour suppressors in the context of cancer and illustrates how oncogenic RAS-mediated degradation of FOXOs, via post-translational mechanisms, blocks these important tumour suppressors.

Protein modifications by the covalent linkage of ubiquitin have significant involvement in many cellular processes, including stress response, oncogenesis, viral infection, transcription, protein turnover, organelle biogenesis, DNA repair, cellular differentiation, and cell cycle control. We provide a brief overview of the fundamentals of the regulation of protein turnover by the ubiquitin-proteasome pathway and discuss new therapeutic strategies that aim to mitigate the deleterious effects of its dysregulation in cancer and other human disease pathophysiology.

Autophagy is a tightly controlled self-degradation process utilised by cells to sustain cellular homeostasis and to support cell survival in response to metabolic stress and starvation. Thus, autophagy plays a critical role in promoting cell integrity and maintaining proper function of cellular processes. Defects in autophagy, however, can have drastic implications in human health and diseases, including cancer. Described as a double-edged sword in the context of cancer, autophagy can act as both suppressor and facilitator of tumorigenesis. As such, defining the precise role of autophagy in a multistep event like cancer progression can be complex. Recent findings have implicated a role for components of the autophagy pathway in oncogene-mediated cell transformation, tumour growth, and survival. Notably, aggressive cancers driven by Ras oncoproteins rely on autophagy to sustain a reprogrammed mitochondrial metabolic signature and evade cell death. In this review, we summarize our current understanding of the role of oncogene-induced autophagy in cancer progression and discuss how modulators of autophagic responses can bring about therapeutic benefit and eradication of a subset of cancers that are addicted to this ancient recycling machinery.

Genome-wide association studies have identified genetic markers in kallikrein-related peptidase 3 (KLK3) associated with prostate cancer. However, some of these markers are also associated with prostate-specific antigen (PSA) levels, so it is unclear whether the polymorphisms are causal or if the association with risk is solely due to detection bias through PSA screening. PSA is a biologically active serine protease, cleaving insulin-like growth factor-binding protein. We examined the association of single-nucleotide polymorphisms (SNPs) in KLK3 with prostate cancer risk, disease-specific survival and pre-diagnostic PSA levels in a case–control study nested within the Physicians’ Health Study, which began in 1982, with over 27 years of follow-up. We genotyped SNPs spanning the entire KLK3 locus to capture common variation at high resolution. Six polymorphisms were significantly associated with prostate cancer incidence (P < 0.05); the odds ratios per minor allele ranged from 0.88 to 0.73. For four of these, the odds ratios were lower when restricting to cases diagnosed in the pre-PSA screening era (before 1989). The four alleles significantly associated with lower PSA levels were also associated with lower prostate cancer risk. KLK3 variants were not significantly associated with stage at diagnosis, risk of lethal cancer or survival. Our results suggest that detection bias due to the association of KLK3 variants with PSA levels cannot completely explain the association with prostate cancer risk. Understanding the mechanism by which genetic variation in KLK3 affects prostate cancer risk has important implications for study of the biological role of PSA in prostate tumorigenesis.

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.

Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukemia (CML). However these inhibitors have been less effective as single agents in the terminal blast phase (BP). We show that pyrvinium, a FDA-approved anthelminthic drug, selectively targets BP-CML CD34+ progenitor cells. Pyrvinium is effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients, while cord blood CD34+ are largely unaffected. The effects of pyrvinium are further enhanced upon combination with dasatinib, a second generation BCR-ABL1 TKI. In a CML xenograft model pyrvinium significantly inhibits tumor growth as a single agent, with complete inhibition in combination with dasatinib. While pyrvinium has been shown to inhibit the Wnt/β-catenin signalling pathway via activation of casein kinase 1α , we find its activity in CML is not dependent on this pathway. Instead, we show that pyrvinium localizes to mitochondria and induces apoptosis by inhibiting mitochondrial respiration. Our study suggests that pyrvinium is a useful addition to the treatment armamentarium for BP-CML and that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukemia.

The G1 D-type cyclins, in conjunction with cyclin-dependent kinases Cdk4 and Cdk6, play key roles in the execution of mitogen-induced cellular proliferation. TRIP-Br1, a member of the TRIP-Br family of transcriptional regulators, has been implicated in the regulation of Cdk4/cyclin D activity. To further elucidate the functional role(s) of the TRIP-Br proteins in mitogenic signaling, we have developed the synthetic DNA enzymes E-Br1 and E-Br2 to specifically knock down the serum-inducible expression of TRIP-Br1 and TRIP-Br2, respectively, in WI-38 human fibroblasts in culture, as well as generated TRIP-Br2 null primary embryonic fibroblasts from a novel TRIP-Br2 knockout mouse model. Both strategies consistently reveal that ablation of TRIP-Br1 or TRIP-Br2 expression disrupts mitogenic signaling in a manner that suppresses serum-induced cyclin E expression, S-phase entry and cellular proliferation. We conclude that both TRIP-Br1 and TRIP-Br2 are required for proper transduction of mitogenic signals and execution of serum-inducible cell cycle progression.

Aberrant Wnt signaling has been widely accepted to be a key driver of a subset of human cancers and a heavily scrutinized molecular pathway for the development of personalized medicine. In a recently published issue of Science Translational Medicine , Rosenberg and coworkers reported that the delta isoform of the CK1 family of serine/threonine kinases (CK1δ), an important mediator of intracellular Wnt signaling, is amplified and overexpressed in human breast tumors. They further demonstrated that pharmacological inhibition of CK1δ is efficacious for these cancers and implicate β-catenin signaling as a key target of CK1δ. In this perspective, we will discuss the salient features of this novel anti-cancer therapeutic approach and the challenges that lie ahead to translate it into a viable treatment option for cancer patients.

Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1α), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RAS-induced autophagy and the development of mutant RAS-driven cancers.

Obesity develops as a result of altered energy homeostasis favoring fat storage. Here we describe a new transcription co-regulator for adiposity and energy metabolism, SERTA domain containing 2 (TRIP-Br2, also called SERTAD2). TRIP-Br2-null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of these knockout mice showed greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and β3-adrenergic (Adrb3) receptors. The knockout mice also have higher energy expenditure because of increased adipocyte thermogenesis and oxidative metabolism caused by upregulating key enzymes in their respective processes. Our data show that a cell-cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data, together with the observation that TRIP-Br2 expression is selectively elevated in visceral fat in obese humans, suggests that this transcriptional co-regulator is a new therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.

The CK1 family of serine/threonine kinases regulates diverse cellular processes, through binding to and phosphorylation a myriad of protein substrates. CK1 prefers substrates primed by prior phosphorylation, and works closely with other kinases in the Wnt pathway. CK1 is itself regulated by posttranslational modification, including autophosphorylation. We provide a brief overview of the fundamentals of CK1 biology with an emphasis on scaffold binding and kinase regulation in Wnt signaling and circadian rhythms.

Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A–WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

The use of Bcr-Abl tyrosine kinase inhibitors (TKIs) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukaemia (CML). However these TKIs have been less effective as single agents in blast phase (BP) CML and this represents an urgent unmet need. A number of novel agents are now being investigated but most have not been translated to the clinics yet. Pyrvinium, a FDA-approved anthelminthic drug, was reported to selectively inhibit growth of a number of tumour cell types including myeloma and erythroleukaemia. We investigated the effect and mechanism of action of pyrvinium in BP-CML. Our results show that pyrvinium selectively targeted BP-CML CD34+ progenitor cells. It induced apoptosis in CD34+ cells from TKI-resistant BP-CML patients who harbour Bcr-Abl kinase mutations, while sparing normal cord blood CD34+ cells. In addition, pyrvinium was more effective in inhibiting colony formation and self-renewal capacity in BP-CML CD34+ cells than cord blood CD34+ cells. Further increase in apoptosis, decrease in colony formation and self-renewal were seen when dasatinib was combined with pyrvinium in BP-CML but not cord blood CD34+ cells (Figure 1 A-C). We also showed that pyrvinium was synergistic in combination with dasatinib in inhibiting proliferation and inducing apoptosis in CML cell lines. We next tested the effects of pyrvinium and its combination with dasatinib in a CML xenograft model and showed that pyrvinium significantly delayed tumour growth with no signs of toxicity in the mice. When combined with dasatinib, the tumour growth was completely inhibited (Figure 1D). These results indicate that pyrvinium is active in BP-CML in vitro and in vivo. The anti-cancer effects of pyrvinium have been reported to due to allosteric activation of casein kinase 1α (CK1α) and suppression of Wnt/β-catenin signalling (Thorne et al, Nature Chemical Biology 2010). However we showed that despite the efficient knockdown of CK1α and overexpression of β-catenin, the proliferation inhibitory effect of pyrvinium was not abolished, indicating a CK1α and β-catenin-independent mechanism of action in BP-CML. Instead, we observed that pyrvinium preferentially localized to mitochondria in CML cells and that pyrvinium inhibited oxygen consumption of CML cells within 5 minutes of treatment. In mitochondrial respiratory chain-deficient CML ρ0 cells which lack mitochondrial DNA and have undetectable oxygen consumption, we showed that the effect of pyrvinium in reducing ATP levels and inducing apoptosis was abolished. These results indicate that pyrvinium acts in CML through the inhibition of mitochondrial respiration. We have shown that pyrvinium alone and in combination with Bcr-Abl TKI selectively targets BP-CML progenitor cells and pyrvinium acts in BP-CML through the inhibition of mitochondrial respiration. Given that pyrvinium is already clinically available, our pre-clinical findings can be translated rapidly into the clinics. Our data also suggests that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukaemia. Figure 1 (A) Pyrvinium induces apoptosis of BP-CML CD34+ but not CB CD34+ cells and combination of pyrvinium and dasatinib is superior in inducing apoptosis. Pyrvinium is more effective in decreasing colony formation (B) and serial replating capacity (C) in BP-CML CD34+ than CB CD34+ cells. The combination is significantly more effective than single drug treatment (* p Disclosures Off Label Use: The use of pyrvinium in leukaemia. Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Casein kinase 1 delta (CK1δ) and CK1 epsilon (CK1ε) regulate Wnt/β catenin signaling and their dysregulation has been implicated in a variety of human cancers. Kinome RNAi screens and as well as targeted studies have identified CK1δ and CK1ε as prosurvival factors in a variety of cancer cell lines. While RNAi knockdown of CK1δ or CK1ε displayed only modest growth inhibition of various cell lines, the CKIδ/ε selective inhibitor 3-[2,4,6-(trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261)(with an IC50 in the 20–50 µM range) has proven even more effective at selective killing of cancer cells, even at submicromolar levels. This finding has been taken as supportive evidence for the role of CKIε and CKIδ in cancer. The recent development of a selective and nanomolar CKIδ/ε inhibitor, PF670462, affords an opportunity to further explore this hypothesis. We found that PF670462 effectively blocked CK1 kinase activity and suppressed Wnt/β-catenin signaling in both HEK293 cells and HT1080 fibrosarcoma cells at submicromolar concentrations. However, consistent with the results of siRNA knockdown of CK1ε, PF670462 failed to induce cancer cell death at these concentrations. In contrast, we found that while 100 nM IC261 did not inhibit of CK1ε activity nor block Wnt/ catenin signaling in vivo, it resulted in the rapid induction of G2/M arrest of cell cycle and apoptosis in human cancer, but not non-transformed, cells. Taken together, inactivation of CK1δ/ε and subsequent block of Wnt/β catenin signaling may not be sufficient to inhibit cancer cell survival. Furthermore, IC261 potently regulates additional cancer cell targets at concentrations much lower than that required for CK1 inhibition and these novel unknown targets appear to be important for the maintenance of human cancer cell survival. Identification of the IC261 targets may provide insights into the mechanism of selective cancer cell toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B264.