ALBERT

Beschreibung: Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.

Beschreibung: Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustrated by the presence of reticulin fibers stained with silver, mainly in spleen. Presence of fibrosis was not as pronounced in heterozygous del52 mice and more rarely observed in spleen of homozygous ins5 KI mice. In homozygous del52 KI mice, there was a significant amplification of the HSC compartment in both BM and spleen that was stronger than in homozygous ins5 mice. To study whether del52 and ins5 could provide a competitive advantage to HSCs, we performed BM transplantation with increasing percentages of non-induced homozygous del52 or ins5 cells with wild-type GFP+ cells into lethally-irradiated recipient mice. The homozygous del52 BM cells strongly competed wild-type hematopoiesis from an initial engraftment as low as 10% of mutated clones, reaching 100% in both blood myeloid cells and BM HSC compartments at 4 months. In contrast, out-competition of wild-type hematopoiesis by homozygous ins5 cells was slower, especially when less than 50% of mutated cells were initially engrafted suggesting that del52 provides a stronger advantage to the HSCs than ins5. Conclusion In conclusion, these results demonstrate that modeling CALRdel52 and ins5 mutations in mice can successfully recapitulate the differences in phenotype observed in patients, i.e del52 KI mice recapitulate an ET progressing to MF while ins5 KI mice only mimic an ET. This might be explained by a more profound effect of del52 than ins5 at the level of HSC. These KI mice offer solid in vivo models to investigate the mechanism of action of both types of mutations on HSCs and MKs and will be used to test new therapeutic approaches. Disclosures: No relevant conflicts of interest to declare. Disclosures Constantinescu: AlsaTech: Other: Co-Founde; Wiley & Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics.

Beschreibung: Introduction Primary and secondary myelofibrosis (MF) present the worst prognosis in the group of BCR-ABL-negative myeloproliferative neoplasms. Presently there is no really efficient therapy of myelofibrosis, except allogenic bone marrow transplantation. In PMF patients, the JAK inhibitors ruxolitinib and fedratinib do not significantly reduce the MF itself or the natural progression of the disease. This could be due to the absence of direct link between JAK2 signaling level and the development of MF. However, ruxolitinib only partially inhibits JAK2 signaling and a reactivation of JAK/STAT signaling through heterodimerization of JAK2 with other JAK may occur (Koppikar et al, Nature, 2012). Complete inhibition of JAK2 signaling would likely induce a profound anemia and thrombocytopenia and at long-term an aplastic anemia. It therefore remains of great interest to identify and inhibit specific downstream targets dysregulated by JAK2 activation that may be responsible of the MF to circumvent the absence of a specific JAK2V617F inhibitor. JAK2V617F mutation leads to the autonomous activation of homodimeric type 1 cytokine receptors and their downstream signaling that induces G1/S cell cycle transition by activating the cyclin-dependent kinases CDK4/6. Cdk6 loss in Jak2V617F KI mice delays the development of the MPN and increases mouse survival (Uras et al, Blood, 2019). We hypothesized that intensifying the JAK2V617F-mediated CDK4/6 increased activity could potentially accelerate the MF development, which is a late event in the Jak2V617F KI model. Our study aims to explore the role of CDK4/6 overactivation or inhibition in the disease and MF development. Methods Since p19INK4D is a cyclin dependent kinase inhibitor, which inhibits CDK4 and to a lesser extent CDK6 and which specifically regulate megakaryocyte (MK) cell cycle and the arrest of endomitosis, we established a pre-clinical MF model by crossing p19Ink4d knock-out (KO) mice with heterozygous inducible Jak2V617F KI mice to obtain p19Ink4d-/-/Jak2FLEXV617F/+SCL-Cre+/-mice called here KOKI mice. With this model, we studied the development of the myeloproliferative disorder. To prevent a MF development in this model, a CDK4/6 inhibitor Palbociclib was administered from 2 to 4 weeks post-Jak2V617F induction. Results In contrast to Jak2V617F (KI) mice that developed a severe MF, in both bone marrow and spleen, at 24 weeks post-Jak2V617F induction, all KOKI mice developed a grade I MF as early as 3 weeks after Jak2V617F induction that progressed to severe fibrosis at 8 weeks (grade III). KOKI mice were characterized by the rapid onset of an extramedullary hematopoiesis (EMH) and megakaryocytic hyperplasia in bone marrow and spleen. As MK are known to be a major source of TGF-β1 in the development of MF, we further focused on the MK lineage. At 1 week post-Jak2V617F induction, prior to MF development, the numbers of MK progenitors (MK-P) were markedly increased in the spleen of KOKI mice (11-fold) while only 3.9-fold increase was observed for KI mice as compared to WT littermates. A significant 2.6-fold increase in the number of MK in the spleen was only visible in KOKI mice, in accordance with splenomegaly and EMH. The analysis of mean ploidy level (〉4N) revealed that it increased to 34.8N in KI, 43.7N in KO p19 and 59.4N in KOKI mice as compared to 19N in WT mice. Total TGF-β1 bone marrow levels were moderately elevated in KOKI mice, with a higher proportion of activated TGF-β1. Treatment of KOKI mice with a CDK4/6 inhibitor, Palbociclib, prevented the development of MF and markedly decreased the EMH, the abnormalities of the megakaryocyte differentiation including the ploidy level and the total and activated TGF-β1 levels. Conclusions Altogether, using a KOKI mouse model we show that the deregulation of CDK4/6 mainly in megakaryocytes is sufficient to induce a severe MF strongly suggesting that CDK4/6 inhibitors could be a very promising therapeutic issue for MF patients (Figure). Disclosure of Prior Presentation/Publication : The abstract will not have been presented and published between January 1, 2020 and December 5, 2020. Figure 1 Disclosures No relevant conflicts of interest to declare.

Beschreibung: ω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.