Fechter, L. | Search bioRxiv

bioRxiv2023

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR by shRNA in CD34+ derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/523111v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@80834corg.highwire.dtl.DTLVardef@12c033forg.highwire.dtl.DTLVardef@8948c8org.highwire.dtl.DTLVardef@1096184_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIEnkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs. C_LIO_LIMPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20. C_LIO_LILikely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. C_LI

cancer biology↗

bioRxiv2021

Progressive and predictive markers of disease evolution: platelet transcriptome in chronic myeloproliferative neoplasms

Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs) as a model, we identify the blood platelet transcriptome as a proxy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine learning algorithms. Using RNA sequencing (RNA-seq), we derive disease-relevant gene expression in purified platelets from 120 peripheral blood samples constituting two time-separated cohorts of patients diagnosed with one of three MPN subtypes at sample acquisition - essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), and healthy donors (n=21). The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy and discriminates each clinical phenotype. Leveraging this dataset that shows a characteristic progressive expression gradient across MPN, we develop a machine learning model (Lasso-penalized regression) and predict advanced subtype MF at high accuracy and under two conditions of external validation: i) temporal: our two Stanford cohorts, AUC-ROC of 0.96; and ii) geographical: independently published data of an additional n=25 MF and n=46 healthy donors, AUC-ROC of 0.97). Lasso-derived signatures offer a robust core set of < 5 MPN transcriptome markers that are progressive in expression. Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with persistent integrated stress response. We also identify JAK inhibitor-specific signatures and other interferon, proliferation, and proteostasis-associated markers as putative targets for MPN-directed therapy. Our platelet transcriptome snapshot of chronic MPNs demonstrates a proof of principle for disease risk stratification and progression beyond genetic data alone, with potential utility in other progressive disorders. HighlightsLeveraging two independent and mutually validating MPN patient cohorts, we identify progressive transcriptomic markers that also enable externally validated prediction in MPNs. Our platelet RNA-Seq data identifies impaired protein homeostasis as prominent in MPN progression and offers putative targets of therapy. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/435190v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@72dc16org.highwire.dtl.DTLVardef@cf5096org.highwire.dtl.DTLVardef@b3d460org.highwire.dtl.DTLVardef@3c0ddc_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗