Identification of key microRNAs as predictive biomarkers of Nilotinib response in chronic myeloid leukemia: a sub-analysis of the ENESTxtnd clinical trial.

Despite the effectiveness of tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML), they are not usually curative as some patients develop drug-resistance or are at risk of disease relapse when treatment is discontinued. Studies have demonstrated that primitive CML cells display unique miRNA profiles in response to TKI treatment. However, the utility of miRNAs in predicting treatment response is not yet conclusive. Here, we analyzed differentially expressed miRNAs in CD34+ CML cells pre- and post-nilotinib (NL) therapy from 58 patients enrolled in the Canadian sub-analysis of the ENESTxtnd phase IIIb clinical trial which correlated with sensitivity of CD34+ cells to NL treatment in in vitro colony-forming cell (CFC) assays. We performed Cox Proportional Hazard (CoxPH) analysis and applied machine learning algorithms to generate multivariate miRNA panels which can predict NL response at treatment-naïve or post-treatment time points. We demonstrated that a combination of miR-145 and miR-708 are effective predictors of NL response in treatment-naïve patients whereas miR-150 and miR-185 were significant classifiers at 1-month and 3-month post-NL therapy. Interestingly, incorporation of NL-CFC output in these panels enhanced predictive performance. Thus, this novel predictive model may be developed into a prognostic tool for use in the clinic.

Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia.

The abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

Puncta intended: connecting the dots between autophagy and cell stress networks.

Proteome profiling and global protein-interaction approaches have significantly improved our knowledge of the protein interactomes of autophagy and other cellular stress-response pathways. New discoveries regarding protein complexes, interaction partners, interaction domains, and biological roles of players that are part of these pathways are emerging. The fourth Vancouver Autophagy Symposium showcased research that expands our understanding of the protein interaction networks and molecular mechanisms underlying autophagy and other cellular stress responses in the context of distinct stressors. In the keynote presentation, Dr. Wade Harper described his team's recent discovery of a novel reticulophagy receptor for selective autophagic degradation of the endoplasmic reticulum, and discussed molecular mechanisms involved in ribophagy and non-autophagic ribosomal turnover. In other presentations, both omic and targeted approaches were used to reveal molecular players of other cellular stress responses including amyloid body and stress granule formation, anastasis, and extracellular vesicle biogenesis. Additional topics included the roles of autophagy in disease pathogenesis, autophagy regulatory mechanisms, and crosstalk between autophagy and cellular metabolism in anti-tumor immunity. The relationship between autophagy and other cell stress responses remains a relatively unexplored area in the field, with future investigations required to understand how the various processes are coordinated and connected in cells and tissues.Abbreviations: A-bodies: amyloid bodies; ACM: amyloid-converting motif; AMFR/gp78: autocrine motility factor receptor; ATG: autophagy-related; ATG4B: autophagy related 4B cysteine peptidase; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CAR T: chimeric antigen receptor T; CASP3: caspase 3; CCPG1: cell cycle progression 1; CAR: chimeric antigen receptor; CML: chronic myeloid leukemia; CCOCs: clear cell ovarian cancers; CVB3: coxsackievirus B3; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9; DDXs: DEAD-box helicases; EIF2S1/EIF-2alpha: eukaryotic translation initiation factor 2 subunit alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; EV: extracellular vesicle; FAO: fatty acid oxidation; GABARAP: GABA type A receptor-associated protein; ILK: integrin linked kinase; ISR: integrated stress response; MTOR: mechanistic target of rapamycin kinase; MPECs: memory precursory effector T cells; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; PI4KB/PI4KIIIß: phosphatidylinositol 4-kinase beta; PLEKHM1: pleckstrin homology and RUN domain containing M1; RB1CC1: RB1 inducible coiled-coil 1; RTN3: reticulon 3; rIGSRNAs: ribosomal intergenic noncoding RNAs; RPL29: ribosomal protein L29; RPS3: ribosomal protein S3; S. cerevisiae: Saccharomyces cerevisiae; sEV: small extracellular vesicles; S. pombe: Schizosaccharomyces pombe; SQSTM1: sequestosome 1; SF3B1: splicing factor 3b subunit 1; SILAC-MS: stable isotope labeling with amino acids in cell culture-mass spectrometry; SNAP29: synaptosome associated protein 29; TEX264: testis expressed 264, ER-phagy receptor; TNBC: triple-negative breast cancer; ULK1: unc-51 like autophagy activating kinase 1; VAS: Vancouver Autophagy Symposium.

Loss of m1acp3Ψ Ribosomal RNA Modification Is a Major Feature of Cancer.

The ribosome is an RNA-protein complex that is essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We identify a cancer-specific single-nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of patients with colorectal carcinoma (CRC) and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine (m1acp3Ψ), a >1-billion-years-conserved RNA modification at the peptidyl decoding site of the ribosome. A subset of CRC tumors we call hypo-m1acp3Ψ shows sub-stoichiometric m1acp3Ψ modification, unlike normal control tissues. An m1acp3Ψ knockout model and hypo-m1acp3Ψ patient tumors share a translational signature characterized by highly abundant ribosomal proteins. Thus, m1acp3Ψ-deficient rRNA forms an uncharacterized class of "onco-ribosome" which may serve as a chemotherapeutic target for treating cancer patients.

Integrin-Linked Kinase Mediates Therapeutic Resistance of Quiescent CML Stem Cells to Tyrosine Kinase Inhibitors.

Patients with chronic myeloid leukemia (CML) often require lifelong therapy with ABL1 tyrosine kinase inhibitors (TKIs) due to a persisting TKI-resistant population of leukemic stem cells (LSCs). From transcriptome profiling, we show integrin-linked kinase (ILK), a key constituent of focal adhesions, is highly expressed in TKI-nonresponsive patient cells and their LSCs. Genetic and pharmacological inhibition of ILK impaired the survival of nonresponder patient cells, sensitizing them to TKIs, even in the presence of protective niche cells. Furthermore, ILK inhibition eliminated TKI-refractory LSCs from patients, but not normal HSCs, in vitro and in vivo. RNA-sequencing and functional validation studies implicated an important role of ILK in maintaining a requisite level of mitochondrial oxidative metabolism in highly purified, quiescent LSCs. Thus, these findings point to ILK as a critical survival mediator to TKIs and quiescent stem cells, offering an attractive therapeutic target and model for curative combination therapies in stem-cell-driven cancers.

The miR-185/PAK6 axis predicts therapy response and regulates survival of drug-resistant leukemic stem cells in CML.

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.

Response to Comment on "PP2A inhibition sensitizes cancer stem cells to ABL tyrosine kinase inhibitors in BCR-ABL+ human leukemia".

LB100 sensitizes resistant chronic phase CML stem and progenitor cells to TKIs and spares healthy bone marrow cells.

Current Outlook on Autophagy in Human Leukemia: Foe in Cancer Stem Cells and Drug Resistance, Friend in New Therapeutic Interventions.

Autophagy is an evolutionarily conserved cellular recycling process in cell homeostasis and stress adaptation. It confers protection and promotes survival in response to metabolic/environmental stress, and is upregulated in response to nutrient deprivation, hypoxia, and chemotherapies. Autophagy is also known to sustain malignant cell growth and contributes to cancer stem cell survival when challenged by cytotoxic and/or targeted therapies, a potential mechanism of disease persistence and drug resistance that has gathered momentum. However, different types of human leukemia utilize autophagy in complex, context-specific manners, and the molecular and cellular mechanisms underlying this process involve multiple protein networks that will be discussed in this review. There is mounting preclinical evidence that targeting autophagy can enhance the efficacy of cancer therapies. Chloroquine and other lysosomal inhibitors have spurred initiation of clinical trials and demonstrated that inhibition of autophagy restores chemosensitivity of anticancer drugs, but with limited autophagy-dependent effects. Intriguingly, several autophagy-specific inhibitors, with better therapeutic indexes and lower toxicity, have been developed. Promising preclinical studies with novel combination approaches as well as potential challenges to effectively eradicate drug-resistant cells, particularly cancer stem cells, in human leukemia are also detailed in this review.

Diverse mechanisms of autophagy dysregulation and their therapeutic implications: does the shoe fit?

In its third edition, the Vancouver Autophagy Symposium presented a platform for vibrant discussion on the differential roles of macroautophagy/autophagy in disease. This one-day symposium was held at the BC Cancer Research Centre in Vancouver, BC, bringing together experts in cell biology, protein biochemistry and medicinal chemistry across several different disease models and model organisms. The Vancouver Autophagy Symposium featured 2 keynote speakers that are well known for their seminal contributions to autophagy research, Dr. David Rubinsztein (Cambridge Institute for Medical Research) and Dr. Kay F. Macleod (University of Chicago). Key discussions included the context-dependent roles and mechanisms of dysregulation of autophagy in diseases and the corresponding need to consider context-dependent autophagy modulation strategies. Additional highlights included the differential roles of bulk autophagy versus selective autophagy, novel autophagy regulators, and emerging chemical tools to study autophagy inhibition. Interdisciplinary discussions focused on addressing questions such as which stage of disease to target, which type of autophagy to target and which component to target for autophagy modulation. Abbreviations: AD: Alzheimer disease; AMFR/Gp78: autocrine motility factor receptor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CML: chronic myeloid leukemia; CVB3: coxsackievirus B3; DRPLA: dentatorubral-pallidoluysian atrophy; ER: endoplasmic reticulum; ERAD: ER-associated degradation; FA: focal adhesion; HCQ: hydroxychloroquine; HD: Huntingtin disease; HIF1A/Hif1α: hypoxia inducible factor 1 subunit alpha; HTT: huntingtin; IM: imatinib mesylate; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; NBR1: neighbour of BRCA1; OGA: O-GlcNAcase; PDAC: pancreatic ductal adenocarcinoma; PLEKHM1: pleckstrin homology and RUN domain containing M1; polyQ: poly-glutamine; ROS: reactive oxygen species; RP: retinitis pigmentosa; SNAP29: synaptosome associated protein 29; SPCA3: spinocerebellar ataxia type 3; TNBC: triple-negative breast cancer.

PP2A inhibition sensitizes cancer stem cells to ABL tyrosine kinase inhibitors in BCR-ABL+ human leukemia.

Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges in curing BCR-ABL+ human leukemia. Using an advanced drug/proliferation screen, we have uncovered a prosurvival role for protein phosphatase 2A (PP2A) in tyrosine kinase inhibitor (TKI)-insensitive leukemic cells, regulated by an Abelson helper integration site-1-mediated PP2A-ß-catenin-BCR-ABL-JAK2 protein complex. Genetic and pharmacological inhibition of PP2A impairs survival of TKI nonresponder cells and sensitizes them to TKIs in vitro, inducing a dramatic loss of several key proteins, including ß-catenin. We also demonstrate that the clinically validated PP2A inhibitors LB100 and LB102, in combination with TKIs, selectively eliminate treatment-naïve TKI-insensitive stem and progenitor cells, while sparing healthy counterparts. In addition, PP2A inhibitors and TKIs act synergistically to inhibit the growth of TKI-insensitive cells, as assessed by combination index analysis. The combination eliminates infiltrated BCR-ABL+ blast cells and drug-insensitive LSCs and confers a survival advantage in preclinical xenotransplant models. Thus, dual PP2A and BCR-ABL inhibition may be a valuable therapeutic strategy to synergistically target drug-insensitive LSCs that maintain minimal residual disease in patients.

CuboCube: Student creation of a cancer genetics e-textbook using open-access software for social learning.

Student creation of educational materials has the capacity both to enhance learning and to decrease costs. Three successive honors-style classes of undergraduate students in a cancer genetics class worked with a new software system, CuboCube, to create an e-textbook. CuboCube is an open-source learning materials creation system designed to facilitate e-textbook development, with an ultimate goal of improving the social learning experience for students. Equipped with crowdsourcing capabilities, CuboCube provides intuitive tools for nontechnical and technical authors alike to create content together in a structured manner. The process of e-textbook development revealed both strengths and challenges of the approach, which can inform future efforts. Both the CuboCube platform and the Cancer Genetics E-textbook are freely available to the community.

Precision autophagy: Will the next wave of selective autophagy markers and specific autophagy inhibitors feed clinical pipelines?

Research presented at the Vancouver Autophagy Symposium (VAS) 2014 suggests that autophagy's influence on health and disease depends on tight regulation and precision targeting of substrates. Discussions recognized a pressing need for robust biomarkers that accurately assess the clinical utility of modulating autophagy in disease contexts. Biomarker discovery could flow from investigations of context-dependent triggers, sensors, and adaptors that tailor the autophagy machinery to achieve target specificity. In his keynote address, Dr. Vojo Deretic (University of New Mexico) described the discovery of a cargo receptor family that utilizes peptide motif-based cargo recognition, a mechanism that may be more precise than generic substrate tagging. The keynote by Dr. Alec Kimmelman (Harvard Medical School) emphasized that unbiased screens for novel selective autophagy factors may accelerate the development of autophagy-based therapies. Using a quantitative proteomics screen for de novo identification of autophagosome substrates in pancreatic cancer, Kimmelman's group discovered a new type of selective autophagy that regulates bioavailable iron. Additional presentations revealed novel autophagy regulators and receptors in metabolic diseases, proteinopathies, and cancer, and outlined the development of specific autophagy inhibitors and treatment regimens that combine autophagy modulation with anticancer therapies. VAS 2014 stimulated interdisciplinary discussions focused on the development of biomarkers, drugs, and preclinical models to facilitate clinical translation of key autophagy discoveries.

Selective JAK2/ABL dual inhibition therapy effectively eliminates TKI-insensitive CML stem/progenitor cells.

Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on the treatment of chronic myeloid leukemia (CML). However, TKI monotherapy is not curative, with relapse and persistence of leukemic stem cells (LSCs) remaining a challenge. We have recently identified an AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance in CML stem/progenitor cells. JAK2 thus emerges as an attractive target for improved therapies, but off-target effects of newly developed JAK2 inhibitors on normal hematopoietic cells remain a concern. We have examined the biological effects of a highly selective, orally bioavailable JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34+ treatment-naïve IM-nonresponder cells. Combination therapy reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits proliferation and colony growth, and eliminates CML LSCs in vitro. Importantly, BMS-911543 selectively targets CML stem/progenitor cells while sparing healthy stem/progenitor cells. Oral BMS-911543 combined with the potent TKI dasatinib more effectively eliminates infiltrated leukemic cells in hematopoietic tissues than TKI monotherapy and enhances survival of leukemic mice. Dual targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may consequently lead to more effective disease eradication, especially in patients at high risk of TKI resistance and disease progression.

The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells.

Previous studies demonstrated that imatinib mesylate (IM) induces autophagy in chronic myeloid leukemia (CML) and that this process is critical to cell survival upon therapy. However, it is not known if the autophagic process differs at basal levels between CML patients and healthy individuals and if pretreatment CML cells harbor unique autophagy characteristics that could predict patients' clinical outcomes. We now demonstrate that several key autophagy genes are differentially expressed in CD34(+) hematopoietic stem/progenitor cells, with the highest transcript levels detected for ATG4B, and that the transcript and protein expression levels of ATG4 family members, ATG5 and BECLIN-1 are significantly increased in CD34(+) cells from chronic-phase CML patients (P < .05). Importantly, ATG4B is differentially expressed in pretreatment CML stem/progenitor cells from subsequent IM responders vs IM nonresponders (P < .05). Knockdown of ATG4B suppresses autophagy, impairs the survival of CML stem/progenitor cells and sensitizes them to IM treatment. Moreover, deregulated expression of ATG4B in CD34(+) CML cells inversely correlates with transcript levels of miR-34a, and ATG4B is shown to be a direct target of miR-34a. This study identifies ATG4B as a potential biomarker for predicting therapeutic response in treatment-naïve CML stem/progenitor cells and uncovers ATG4B as a possible drug target in these cells.

Autophagy: from structure to metabolism to therapeutic regulation.

Multidisciplinary approaches are increasingly being used to elucidate the role of autophagy in health and disease and to harness it for therapeutic purposes. The broad range of topics included in the program of the Vancouver Autophagy Symposium (VAS) 2013 illustrated this multidisciplinarity: structural biology of Atg proteins, mechanisms of selective autophagy, in silico drug design targeting ATG proteins, strategies for drug screening, autophagy-metabolism interplay, and therapeutic approaches to modulate autophagy. VAS 2013 took place at the British Columbia Cancer Research Centre, and was hosted by the CIHR Team in Investigating Autophagy Proteins as Molecular Targets for Cancer Treatment. The program was designed as a day of research exchanges, featuring two invited keynote speakers, internationally recognized for their groundbreaking contributions in autophagy, Dr Ana Maria Cuervo (Albert Einstein College of Medicine, Bronx, NY) and Dr Jayanta Debnath (University of California, San Francisco). By bringing together international and local experts in cell biology, drug discovery, and clinical translation, the symposium facilitated rich interdisciplinary discussions focused on multiple forms of autophagy and their regulation and modulation in the context of cancer.