Key Points Mutant CALR induces TPO-independent growth in the human megakaryocytic cell line UT-7/TPO. Mutant CALR binds to the TPO receptor, inducing phosphorylation of JAK2 and activating downstream signaling.

Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.

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The pseudokinase Trib1 functions as a myeloid oncogene that recruits the E3 ubiquitin ligase COP1 to C/EBPa and interacts with MEK1 to enhance ERK phosphorylation. Close genetic effect of Trib1 on Hoxa9 has been observed in myeloid leukemogenesis where Trib1 overexpression significantly accelerates Hoxa9-induced leukemia onset. However, the mechanism underlying how Trib1 functionally modulates Hoxa9 transcription activity is unclear. Herein, we provide evidence that Trib1 modulates Hoxa9-associated super-enhancers. ChIP-seq analysis identified increased histone H3K27Ac signals at super-enhancers of the Erg, Spns2, Rgl1, and Pik3cd loci, as well as increased mRNA expression of these genes. Modification of super-enhancer activity was mostly achieved via the degradation of C/EBPa p42 by Trib1, with a slight contribution from the MEK/ERK pathway. Silencing of Erg abrogated the growth advantage acquired by Trib1 overexpression, indicating that Erg is a critical downstream target of the Trib1/Hoxa9 axis. Moreover, treatment of acute myeloid leukemia (AML) cells with the BRD4 inhibitor JQ1 showed growth inhibition in a Trib1/Erg-dependent manner both in vitro and in vivo. Upregulation of ERG by TRIB1 was also observed in human AML cell lines, suggesting that Trib1 is a potential therapeutic target of Hoxa9-associated AML. Taken together, our study demonstrates a novel mechanism by which Trib1 modulates chromatin and Hoxa9-driven transcription in myeloid leukemogenesis.

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice. 3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs. Conclusion We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. Disclosures Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino-acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160ng/mL, with a mean of 25.64ng/mL. Plasma mutant CALR is found in complex with soluble Transferrin Receptor 1 (sTFRC) that acts as a carrier protein and increases CALR mutant half-life. Recombinant mutant CALR proteins bound and activated the TpoR on cell lines and primary megakaryocytic progenitors from CALR-mutated patients where they drive Tpo-independent colony formation. Importantly, the CALR-sTFRC complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in one cell can specifically interact in trans with the TpoR on a target cell. Cells that carry both TpoR and mutant CALR are hypersensitive to exogenous CALR mutant proteins in comparison to cells that only carry TpoR, and respond to levels of CALR mutant proteins similar to those in patient plasma. This is consistent with CALR mutated cells exposing at the cell-surface TpoR carrying immature N-linked sugars. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, is frequently detected in classical myeloproliferative neoplasms (MPNs). In the present study, we determined the JAK2V617F allele burden in Japanese MPN patients using alternately binding probe competitive-polymerase chain reaction, a highly quantitative method recently developed by our group. Although we observed strong similarities in terms of epidemiological parameters associated with the JAK2V617F allele burden between our cohort and others, we found a higher JAK2V617F allele burden in Japanese polycythemia vera (PV) patients and lower frequencies of thrombosis in Japanese MPN patients compared with previous reports. In addition, despite the presence of high red blood cell counts, some patients bearing the JAK2V617F mutation were not diagnosed as PV, as their hemoglobin values were lower than the WHO PV criterion. In these patients, the JAK2V617F allele burden was strikingly similar to that in PV patients fulfilling the 2008 WHO criteria, suggesting that these patients can be classified as PV. Although isotopic measurement of red cell mass (RCM) is required for definitive diagnosis of PV, our data suggest that precise measurement of the JAK2V617F allele burden may improve the diagnosis of PV when RCM has not been determined.