A) Expression, in CD34+ cells of PMF patients and controls, of miRNAs and moRNAs produced from the same hairpin, considering the hairpins expressing most abundant moRNAs; the boxplot in panel (B) shows the distribution of Pearson correlation values calculated pairwise between expression profiles of moRNAs and miRNAs produced from the same hairpin arm, considering all moRNAs detected in CD34+ cells.

<p>A) Expression, in CD34+ cells of PMF patients and controls, of miRNAs and moRNAs produced from the same hairpin, considering the hairpins expressing most abundant moRNAs; the boxplot in panel (B) shows the distribution of Pearson correlation values calculated pairwise between expression profiles of moRNAs and miRNAs produced from the same hairpin arm, considering all moRNAs detected in CD34+ cells.</p

Differential expression of small RNAs in PMF vs CTR CD34+.

<p>A) Log2 FC of 37 small RNA differentially expressed considering PMF vs CTR CD34+, according to RNA-seq data (FDR<0.05). When a small RNA was not expressed in one sample category, the ratio was infinite and we represent it as the arbitrary maximum value of 15. B) RT-PCR expression calculation of five selected miRNAs in granulocytes collected from an independent cohort of normal controls (n = 10) and of PMF (50) samples; ***, ** and * indicate respectively a p-value <0.001, <0.01 or <0.05.</p

List of most abundant moRNAs in considered CD34+ samples, which are expressed over the third quartile of all sRNAs expression.

<p>For each moRNA, the table reports expression (per sample group normalized read count), position and sequence.</p

Summary of small RNAs expressed in CD34+ cells.

<p>Summary of small RNAs expressed in CD34+ cells.</p

Differential expression of 3’-moR-128-2 in PMF (n = 3) vs CTR (3) cells.

<p>A) moR expression in PMF and CTR CD34+ according to RNA-seq data. B) RT-PCR expression (RQ) in CD34+ cells from independent cohort of normal controls (n = 8) and of PMF (20) samples. C) RT-PCR expression (RQ) in granulocytes from independent cohort of normal controls (n = 10) and of PMF (50) samples; ***, ** and * indicate respectively a p-value <0.001, <0.01 or <0.05.</p

Origin, sequence variability, and relations beween 3’-moR-128-2 and the adjacent miR-128-3p.

<p>A) 3’-moR-128-2 and miR-128-3p map to the same locus and both shows sequence variability (isomiRs and isomoRs). Both the major and the minor isomoRs are found in normal CD34+ cells and not in PMF samples. Red and blue colors indicate isomiR and isomoR groups that can be produced with an unique sequence cutting sites. The most expressed isomoR is not associated to the corresponding most expressed isomiR. Moreover, expression levels, in CTR and PMF samples, of isomiRs and isomoRs are poorly correlated intragroup. These observations, point against the moRNA being simply a by-product of the miRNA biogenesis. A similar indication is given by the fact that some abundant isomiRs are not associated to detected isomoR sequences. B) 3’-moR-128-2 and miR-128-3p have different, poorly overlapping, sets of predicted targets. C) 3’-moR-128-2 sequence can stably bind a target site in the 3’UTR of the RAN mRNA, causing post transcriptional silencing.</p

The 3’-moR-128-2 is produced by the precursor sequence of miR-128-3p.

<p>A) The moRNA is derived from a region of the primary miRNA sequence exceeding the canonical hairpin precursor sequence, and it is not exaclty adjactent to the annotated miRNA. B) Minimum free energy (MFE) folding structure, predicted by RNAfold, for the canonical hairpin sequence and for the longer one, from which the moRNA is probably derived. C) Both the considered small RNAs are conserved in evolution through vertebrates.</p

Expression pattern of 18 mRNAs potentially targeted by miRNAs, according to qRT-PCR data.

<p>For each mRNA, miRNA and moRNA, we report if it is increased (I) or decreased (D) in the PMF VS CTR sample comparison; the mRNAs p-values associated to a significant difference are listed; the last column indicates if the expression of observed target mRNA variation is inversely related to the corresponding miRNA or moRNA.</p

Putative targets of 3’-moR-128-2 variants in the “Regulatory RNA” pathway.

<p>Putative targets of 3’-moR-128-2 variants in the “Regulatory RNA” pathway.</p

mTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms

<div><h3>Background</h3><p>Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with <em>JAK2</em>V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects <em>in vitro</em> of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.</p> <h3>Findings</h3><p>Mouse and human <em>JAK2</em>V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of <em>JAK2</em>V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.</p> <h3>Conclusions/Significance</h3><p>These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.</p> </div