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Estimating the impact of differential adherence on the comparative effectiveness of stool-based colorectal cancer screening using the CRC-AIM microsimulation model

BackgroundReal-world adherence to colorectal cancer (CRC) screening strategies is imperfect. The CRC-AIM microsimulation model was used to estimate the impact of imperfect adherence on the relative benefits and burdens of guideline-endorsed, stool-based screening strategies. MethodsPredicted outcomes of multi-target stool DNA (mt-sDNA), fecal immunochemical tests (FIT), and high-sensitivity guaiac-based fecal occult blood tests (HSgFOBT) were simulated for 40-year-olds free of diagnosed CRC. For robustness, imperfect adherence was incorporated in multiple ways and with extensive sensitivity analysis. Analysis 1 assumed adherence from 0%-100%, in 10% increments. Analysis 2 longitudinally applied real-world first-round differential adherence rates (base-case imperfect rates=40% annual FIT vs 34% annual HSgFOBT vs 70% triennial mt-sDNA). Analysis 3 randomly assigned individuals to receive 1, 5, or 9 lifetime (9=100% adherence) mt-sDNA tests and 1, 5, or 9 to 26 (26=100% adherence) FIT tests. Outcomes are reported per 1000 individuals compared with no screening. ResultsEach screening strategy decreased CRC incidence and mortality versus no screening. In individuals screened between ages 50-75 and adherence ranging from 10%-100%, the life-years gained (LYG) for triennial mt-sDNA ranged from 133.1-300.0, for annual FIT from 96.3-318.1, and for annual HSgFOBT from 99.8-320.6. At base-case imperfect adherence rates, mt-sDNA resulted in 19.1% more LYG versus FIT, 25.4% more LYG versus HSgFOBT, and generally had preferable efficiency ratios while offering the most LYG. Completion of at least 21 FIT tests is needed to reach approximately the same LYG achieved with 9 mt-sDNA tests. ConclusionsAdherence assumptions affect the conclusions of CRC screening microsimulations that are used to inform CRC screening guidelines. LYG from FIT and HSgFOBT are more sensitive to changes in adherence assumptions than mt-sDNA because they require more tests be completed for equivalent benefit. At imperfect adherence rates, mt-sDNA provides more LYG than FIT or HSgFOBT at an acceptable tradeoff in screening burden.

cancer biology

Capitalizing on paradoxical activation of the MAPK pathway for treatment of Imatinib-resistant mast cell leukemia and chronic myelogenous leukemia

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation". Here we re-analyzing the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KITV560G,D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSA KITD816V and in KIT D816V hematopoietic progenitors obtained from in patient-derived induced pluripotent stem cells (iPS cells). In conclusion, mutated KIT-driven Imatinib resistance can be efficiently bypassed by a low concentration combination of the TKI Ponatinib and the MEK inhibitor Trametinib, potentially reducing the negative side effects associated to MCL therapy.

cancer biology

Impact of Screening and Follow-up Colonoscopy Adenoma Sensitivity on Colorectal Cancer Screening Outcomes in the CRC-AIM Microsimulation Model

BackgroundReal-world data for patients with positive colorectal cancer (CRC) screening stool-tests demonstrates that adenoma detection rates are lower when endoscopists are blinded to the stool-test results. This suggests adenoma sensitivity may be lower for screening colonoscopy than for follow-up to a known positive stool-based test. Previous CRC microsimulation models assume identical sensitivities between screening and follow-up colonoscopies after positive stool-tests. The Colorectal Cancer and Adenoma Incidence and Mortality Microsimulation Model (CRC-AIM) was used to explore the impact on screening outcomes when assuming different adenoma sensitivity between screening and combined follow-up/surveillance colonoscopies. MethodsModeled screening strategies included colonoscopy every 10 years, triennial multitarget stool DNA (mt-sDNA), or annual fecal immunochemical test (FIT) from 50-75 years. Outcomes were reported per 1,000 individuals without diagnosed CRC at age 40. Base-case adenoma sensitivity values were identical for screening and follow-up/surveillance colonoscopies. Ranges of adenoma sensitivity values for colonoscopy performance were developed using different slopes of odds ratio adjustments and were designated as small, medium, or large impact scenarios. ResultsAs the differences in adenoma sensitivity for screening versus follow-up/surveillance colonoscopies became greater, life-years gained (LYG) and reductions in CRC-related incidence and mortality versus no screening increased for mt-sDNA and FIT and decreased for screening colonoscopy. The LYG relative to screening colonoscopy reached >90% with FIT in the base-case scenario and with mt-sDNA in a "medium impact" scenario. ConclusionsAssuming identical adenoma sensitivities for screening and follow-up/surveillance colonoscopies underestimates the potential benefits of stool-based screening strategies.

cancer biology

A JAK/STAT-Mediated Inflammatory Signaling Cascade Drives Oncogenesis In AF10-Rearranged AML

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of AML driven by the most common AF10 fusion proteins, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a comprehensive map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent anti-oncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias. STATEMENT OF SIGNIFICANCEGene fusions of AF10/MLLT10 are recurrent in acute myeloid and lymphoid leukemia and are associated with extremely poor survival outcomes. We show that the JAK1 kinase is required for activation of the AF10 fusion oncotranscriptome and for leukemogenesis. Since a number of JAK/STAT pathways inhibitors are in clinical development or approved for use, our studies may help develop a therapeutic strategy for AF10-rearranged leukemias.

cancer biology

PRL and DNA damage alter the immune response to breast cancer cells

There are conflicting reports on the role of prolactin (PRL) in breast cancer, and its role within the context of the tumour microenvironment is not well understood. In our previous study, we demonstrated a cross-talk between the ataxia telangiectasia-mutated (ATM) DNA damage response pathway and the PRL-Janus-kinase-2 (JAK2)-signal transducer and activator of transcription-5 (STAT5)-heat shock protein-90 (HSP90) pathway. To investigate the role of PRL in tumour initiation and the effect of DNA damage in vivo, we used a model of breast cancer initiation that assesses the ability of breast cancer cells to initiate orthotopic xenograft tumour formation after DNA damage. Breast cancer cells engineered to secrete human PRL or the control cells, were treated with the DNA damaging agent doxorubicin or vehicle and injected into the mammary fat pad of immune-deficient SCID mice. PRL secretion from human breast cancer cells did not change the tumour latency compared to controls, although combined doxorubicin and PRL treatment increased tumour latency. Depletion of glycolipid asialo ganglioside-GM1 positive immune cells using anti-asialo GM1 antibody resulted in faster tumour formation only in the PRL-secreting breast cancer cells that were pre-treated with doxorubicin, and not in the PRL-only or empty vector controls. Additionally, doxorubicin plus PRL treatment of breast cancer cells were shown in vitro to attract cytotoxic NK cells compared to controls, and that this was dependent on the PRLR. These results may shed light on the conflicting reports of PRL in breast cancer and demonstrate that combined breast cancer cell DNA damage and PRL exposure results in anti-tumour activity of asialo-GM1-positive immune cells.

cancer biology

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the lungs with few treatment options. We recently reported loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptors 4 (DR4) and 5 (DR5). Using tissue microarray (TMAs) of tumour samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptors. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wild-type BAP1, but not catalytically-inactive mutant BAP1, reduced promoter activities of DR4 and DR5, suggesting deubiquinase activity plays an important role in the regulation of gene expression. Co-IP studies demonstrated direct binding of BAP1 and the transcription factor Ying Yang 1 (YY1) and ChIP assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Notably, shRNA knockdown of YY1 also increased DR4 and DR5 expression, and sensitivity to TRAIL. These results demonstrate that BAP1 and YY1 together negatively regulate transcriptional activity of TRAIL receptors. BAP1 and YY1 may both therefore be strong therapeutic targets to enhance the efficacy of TRAIL-induced apoptosis. Statement of significanceWe describe how the most-frequently mutated tumour suppressor gene in mesothelioma regulates the response to TNF-related apoptosis-inducing ligand (TRAIL). These findings will accelerate a biomarker-driven cancer therapy.

cancer biology

CRISPR screens in physiologic medium reveal conditionally essential genes in human cells

Forward genetic screens across hundreds of diverse cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly resemble metabolite availability in human blood. To explore how medium composition influences gene essentiality, we performed CRISPR-based screens of human cancer cell lines cultured in traditional versus human plasma-like medium (HPLM). Sets of medium-dependent fitness genes span several cellular processes and can vary with both natural cell-intrinsic diversity and the specific combination of basal and serum components that comprise typical culture media. Notably, we traced the causes for each of three conditional growth phenotypes to the availability of metabolites uniquely defined in HPLM versus traditional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.

cancer biology

The cellular genomic diversity, regulatory states and networking of the metastatic colorectal cancer microenvironment

PurposeThe liver is the most frequent metastatic site for colorectal cancer (CRC). Its microenvironment is modified to provide a niche that allows CRC cell growth. This study focused on characterizing the cellular changes in the metastatic CRC (mCRC) liver tumor microenvironment (TME). Experimental DesignWe analyzed a series of microsatellite stable (MSS) mCRCs to the liver, paired normal liver tissue and peripheral blood mononuclear cells using single cell RNA-seq (scRNA-seq). We validated our findings using multiplexed spatial imaging and bulk gene expression with cell deconvolution. ResultsWe identified TME-specific SPP1-expressing macrophages with altered metabolism features, foam cell characteristics and increased activity for extracellular matrix (ECM) organization. SPP1+ macrophages and fibroblasts expressed complementary ligand receptor pairs with the potential to mutually influence their gene expression programs. TME lacked dysfunctional CD8 T cells and contained regulatory T cells, indicative of immunosuppression. Spatial imaging validated these cell states in the TME. Moreover, TME macrophages and fibroblasts had close spatial proximity, a requirement for intercellular communication and networking. In an independent cohort of mCRCs in the liver, we confirmed the presence of SPP1+ macrophages and fibroblasts using gene expression data. An increased proportion of TME fibroblasts was associated with worst prognosis in these patients. ConclusionsWe demonstrated that mCRC in the liver is characterized by transcriptional alterations of macrophages in the TME. Intercellular networking between macrophages and fibroblasts supports CRC growth in the immunosuppressed metastatic niche in the liver. These features can be used to target these immune checkpoint resistant MSS tumors. TRANSLATIONAL RELEVANCEThe liver is the commonest site for metastatic colorectal cancer (mCRC). Alterations in the tumor microenvironment (TME) allow metastatic cells to seed the distant liver site and grow. Leveraging single-cell RNA sequencing, we discovered a distinct SPP1+ macrophage cell state with pro-fibrogenic gene expression and altered metabolism. These SPP1+ macrophages communicated with fibroblasts, mutually influencing each others gene expression program. Using spatial imaging, we confirmed proximal colocalization between macrophages and fibroblasts in the mCRC TME, which is required for intercellular communication. These states and intercellular communication promoted immunosuppression in the TME, with a lack of dysfunctional anti-tumor CD8 T cells and prevalence of regulatory T cells. Increased fibroblasts were associated with worst prognosis in an independent patient cohort. Our results identified novel TME features that result in reshaping of the metastatic niche that allows progression of mCRC. These features can be potential targets for mCRC treatment, which is microsatellite stable and resistant to immune checkpoint blockade.

cancer biology

Polygenic mutations model the pleiotropic disease of Fanconi Anemia

Fanconi Anemia (FA) is a prototypic genetic disease signified by heterogeneous phenotypes including cancer, bone marrow failure, short stature, congenital abnormalities, infertility, sub-mendelian birth rate, genome instability and high cellular sensitivity to cancer therapeutics1-4. Clinical diagnosis is confirmed by identifying biallelic, homo- or hemizygous mutations in any one of twenty-three FANC genes1,5. Puzzlingly, inactivation of one single Fanc gene in mice fails to faithfully model the human disease manifestations6-8. We here delineate a preclinical Fanc mouse model with mutations in two genes, Fancd1/Brca2 and Fanco/Rad51c, that recapitulates the severity and heterogeneity of the human disease manifestations including death by cancer at young age. Surprisingly, these grave phenotypes cannot be explained by the sum of phenotypes seen in mice with single gene inactivation, which are unremarkable. In contrast to expectations from classic epistasis analysis of genetic pathways, the data instead reveal an unexpected functional synergism of polygenic Fanc mutations. Importantly in humans, whole exome sequencing uncovers that FANC co-mutation in addition to the identified inactivating FANC gene mutation is a frequent event in FA patients. Collectively, the data establish a concept of polygenic stress as an important contributor to disease manifestations, with implications for molecular diagnostics.

cancer biology

Many are called but few are chosen - Multiple clonal origins greatly elevate the functional heterogeneity of tumors

Each tumor is usually accepted to be of a single origin from a progenitor cell. The shared evolutionary paths impose a limit on the nature of genetic diversity of the tumor. However, there are also numerous stem cell niches with independent proliferation potentials. To reconcile the contrasting perspectives, we propose a model whereby each tumor is of multiple clonal origins but the most proliferative one would eclipse other minor clones. The detection of the minor clones would entail an extreme scheme of large-number but small-volume sampling. In two cases of colon tumors so sampled, one indeed has 13 independent clones of disparate sizes and even the smaller clones have tens of thousands of cells dispersed non-locally. The other, much larger, tumor has only one prevailing clone that engulfs two tiny patches of minor clones. In both cases, the expanding clone spawns a hierarchy of subclones that resemble vassal states on its wake of expansion. The timing of metastasis can also be mapped to the precise stage of the clonal expansion. In conclusion, multiple independent clones, likely common but difficult to detect, can greatly elevate the non-neutral diversity within a tumor. This much-elevated diversity has many theoretical and clinical implications.

cancer biology

ASO-based PKM Splice-switching Therapy Inhibits Hepatocellular Carcinoma Cell Growth

The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in the Warburg effect, which is characterized by the preference for aerobic glycolysis for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Previously, we developed antisense oligonucleotides (ASOs) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform and induce apoptosis in cultured glioblastoma cells. Here, we explore the potential of ASO-based PKM splice-switching as a targeted therapy for liver cancer. We utilize a more potent lead cEt/DNA ASO and demonstrate that it induces PKM splice-switching and inhibits the growth of cultured hepatocellular-carcinoma (HCC) cells. This PKM isoform switch increases pyruvate-kinase activity and alters glucose metabolism. The lead ASO and a second ASO targeting a non-overlapping site inhibit tumorigenesis in an orthotopic-xenograft HCC mouse model. Finally, a surrogate mouse-specific ASO induces Pkm splice-switching and inhibits HCC growth, without observable toxicity, in a genetic HCC mouse model. These results lay the groundwork for a potential ASO therapy for HCC. Statement of significanceAntisense oligonucleotides are used to force a change in PKM isoform usage in HCC, reversing the Warburg effect and inhibiting tumorigenesis.

cancer biology

eEF2 kinase enhances the expression of PD-L1 by promoting the translation of its mRNA

Emerging advances in cancer therapy have transformed the landscape from conventional therapies towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are game-changers for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that inhibits the elongation stage of protein synthesis, actually promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5-UTR (5-untranslated region) which starts with a non-canonical CUG codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5-region. This, and other roles of eEF2K in cancer cell biology (e.g., in cell survival and migration), may be exploited for the design of future therapeutic strategies.

biochemistry

miR-200 deficiency promotes lung cancer metastasis by activating cancer-associated fibroblasts

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

cancer biology

ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN-response genes

Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over-expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-Flouororuacil and to radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture conditions. We further noticed that alteration of ETV7 expression could significantly affect the population of breast CSCs, measured by CD44+/CD24low cell population and mammosphere formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive genes significantly repressed in cells over-expressing ETV7, which could be responsible for the increase in the breast CSCs population, as this could be partially reverted by the treatment with IFN-{beta}. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could have prognostic value in breast cancer, as low expression of these genes was associated with a worse prognosis. Therefore, we propose a novel role for ETV7 in breast cancer stem cells plasticity and associated resistance to conventional chemotherapy and radiotherapy, which involves the repression of a group of IFN-responsive genes, potentially reversible upon IFN-{beta} treatment. We, therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens, possibly involving the therapeutic use of IFN-{beta}, with the aim of avoiding resistance development and relapse in breast cancer.

cancer biology

Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naive and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities. SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

cancer biology

The lysosomal TRPML1 channel promotes breast cancer survival by supporting mitochondrial function and cellular metabolism

Triple-negative breast cancer (TNBC) is an aggressive subtype representing approximately 10%-20% of breast cancers and lacking effective therapies. TRPML1, which is a lysosomal Ca2+ release channel upregulated in TNBC, promotes TNBC tumor growth. Here we show a novel crosstalk between lysosomes and mitochondria mediated by TRPML1 in TNBC. TRPML1 is required for the maintenance of mitochondrial function and reactive oxygen species (ROS) homeostasis. TRPML1 knockdown inhibits TNBC mitochondrial respiration, glycolysis and ATP production, leading to reduced proliferation, promotion of cell cycle arrest and apoptosis with enhanced global and mitochondrial ROS. Further, TRPML1 downregulation enhances the cytotoxic effect of Doxorubicin in TNBC cells. Our data reveal a hitherto unknown link between lysosomal TRPML1 channels and mitochondrial metabolism and suggest that TRPML1 inhibition in combination with established chemotherapies could be an effective strategy against TNBC tumors.

cancer biology

Only SF3B1 Mutation involving K700E (And Not Other Codons), Independently Predicts Overall Survival in Myelodysplastic Syndromes

BackgroundSF3B1 mutations (SF3B1mut) in myelodysplastic syndromes (MDS) frequently involve codon K700E and have a favorable prognosis. The prognostic effect of non-K700E SF3B1mut is uncertain. MethodsWe analyzed the clinical-pathologic features and outcomes of a single-institutional series of 94 SF3B1mut and 415 SF3B1wt newly diagnosed untreated MDS patients and explored the differences between K700E and non-K700E subgroups. FindingsNinety-four (19%) patients had SF3B1mut: median age, 74 years. Fifty-five (60%) patients carried K700E. Recurrent non-K700E mutations (39, 40%) included R625, H662 and K666. Compared to SF3B1mut K700E, non-K700E patients had a lower median ANC (1{middle dot}8 vs. 2{middle dot}4, p=0{middle dot}005) and were frequently "high" R-IPSS (revised International Prognostic Scoring System) [7(19%) vs. 2(4%), p=0{middle dot}031]. Non-K700E MDS frequently associated with RUNX1 (26% vs. 7%, p=0{middle dot}012) and exclusively with BCOR, IDH2, and SRSF2 mutations. There was no significant difference in karyotype or SF3B1 variant allele frequency. Most ([~]80%) were treated with hypomethylating agents. SF3B1mut had superior overall survival (OS) than SF3B1wt in all MDS categories [not-reached vs. 25{middle dot}2 months, p=0{middle dot}0003], low-grade MDS, and MDS with ring sideroblasts (MDS-RS). Compared to SF3B1wt, SF3B1mut K700E had superior outcomes in all MDS categories (25 months vs. not-reached, p=0{middle dot}0001), low-grade MDS, and MDS-RS, but no significant difference was seen with non-K700E. By multivariate analysis, absence of SF3B1mut K700E (not non-K700E) independently associated with prognosis. InterpretationSF3B1mut MDS show distinct clinical and mutational profiles, with K700E showing a significantly better OS compared to non-K700E mutations and SF3B1wt. Our study highlights the importance of SF3B1 mutation type in MDS risk assessment. Data Sharing StatementThe datasets generated during and/or analyzed during the current study are not publicly available due to patient privacy concerns but are available from the corresponding author on reasonable request. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe designed this study based on the collective evidence from a systematic search of the literature for outcomes of patients MDS with SF3B1 mutations (SF3B1mut) from January 2013 to June 2020. Both the International Working Group for the Prognosis of MDS (IWG-PM) proposal and 2016 revisions to the World Health Organization (WHO) Classification of Myelodysplastic Syndromes recognize SF3B1mut MDS with <5% blasts (or ring sideroblasts >5% for WHO) as a distinct sub-category, in the absence of other unfavorable features. This was largely based on favorable prognostic outcomes, a distinct gene expression profile, and association with ring sideroblasts. However, the natural history of SF3B1mut MDS is heterogeneous. A high proportion of SF3B1 mutations occur within codon K700, leading to large-scale mRNA downregulation due to branch point recognition error, while the rest occur outside of this codon. The downstream functional effects of SF3B1 mutations outside of the K700 codon are unclear. The clinical course of SF3B1mut MDS patients likely depends on the type of SF3B1 mutation and other features such as variant allele frequency, concomitant gene mutations, and karyotype. Until now, the effects of the different types of SF3B1 mutations were largely unknown. Added value of this studyIn this study, we report distinctive clinicopathologic characteristics and outcomes of MDS patients with SF3B1 mutations segregated based on mutation type: K700E vs. non-K700E. We show that [~]40% of SF3B1 mutated MDS patients have non-K700E mutations. Non-K700E SF3B1mut MDS have distinct clinico-pathologic features, such as lower ANC and frequent association with mutations in RUNX1, BCOR, IDH2, and SRSF2. There was no significant difference in karyotype or SF3B1 variant allele frequency. Importantly, K700E SF3B1mut MDS had superior overall survival compared to SF3B1wt, in all MDS, low-grade MDS, and MDS with ring sideroblasts, but no significant difference was seen with non-K700E. By multivariate analysis, absence of SF3B1mut K700E, but not non-K700E, independently associated with prognosis. Implications of all the available evidenceTo the best of our knowledge, this is the first study to report these findings from a single-institutional series of MDS primarily treated with hypomethylating agents. Our study highlights the importance of determining the SF3B1 mutation type in MDS risk assessment. These findings are important in light of the recent FDA approval of luspatercept based on the results of the MEDALIST trial that suggested sustained hematological responses in SF3B1mut MDS patients.

cancer biology

PPM1D is a neuroblastoma oncogene and therapeutic target in childhood neural tumors.

Majority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms. Here we show that p53-regulating PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded by PPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstrated PPM1D genetic dependency in TP53 wild-type neuroblastoma cell lines, and shRNA PPM1D knockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressing PPM1D showed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1-mutations, Pten-deletions and p53-accumulation, adenocarcinomas and PHOX2B-expressing neuroblastomas establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.

cancer biology