Contributions
Abstract: S196
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
In myeloproliferative neoplasms (MPN), somatic driver mutations of calreticulin (CALR), present in up to 30% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), include 52bp deletions (del52) and 5bp insertions (ins5) in exon 9. Mutant CALR acts by firmly binding to the thrombopoietin receptor (MPL), leading to constitutive activation of Janus kinases (e.g. JAK2) and associated downstream signaling. This results in cellular transformation, including enhanced megakaryopoiesis. The mechanism of increased megakaryopoiesis and the role of mutant CALR zygosity has not fully been elucidated to date.
Aims
Establishment of an MPN patient‑derived induced pluripotent stem (iPS) cell model focusing on CALR mutations in megakaryocytic development.
Methods
iPS cells were reprogrammed from PBMCs of MPN patients with CALRdel52 or CALRins5 mutations, or from healthy donors. Since reprogramming of CALR-mutant cells only resulted in homozygous (hom) and heterozygous (het) clones but not CALRwt clones, CALR mutations were repaired using CRISPR/Cas9n gene editing. iPS cells were differentiated into myeloid cells, with a particular focus on megakaryocytes (MKs). Bulk populations were analyzed by flow cytometry, and CD61+ MKs were purified and further analyzed for RNA expression and ultrastructural morphology.
Results
We established an iPS cell model including CALRins5 and del52 hom and het clones and CRISPR-repaired isogenic (WTcr) clones. Homozygous CALRdel52 and CALRins5 iPS clones were found to lack myeloperoxidase (MPO) activity, as has been described for patients with hom CALR mutations, and MPO activity as an indicator of CALR wt protein function was restored in WTcr clones. We established a protocol allowing efficient generation of iPS cell‑derived MKs with characteristics of primary MKs, such as formation of a demarcation membrane system, cytoplasmic pro-platelet protrusions, and MK-typical granules. We demonstrate that hom and het CALRins5- and del52-mutated iPS cells generate a significantly higher number of MKs than related WTcr clones (p<0.05 each). Moreover, MK generation of CALRins5 and del52 cells is TPO-independent in contrast to WTcr and healthy donor clones. Enhanced megakaryopoiesis was due to an accelerated maturation process of CALR‑mutated MKs, since the number of CD42b+ cells among the population of CD61+CD41+ MKs on day 10 was higher in hom (p=0.001[del52], p=0.18[ins5]) and het (p=0.044[ins5]) CALR‑mutated cells vs. WTcr MKs. In contrast, on day 14, the number of CD42+ MKs in CD61+CD41+ population was similar for all genotypes, suggesting that the WTcr MKs had taken longer to mature. Nonetheless, differences in the expression of megakaryocytic genes were observed between genotypes, with an increased MPL expression in CALRins5 MKs compared to WTcr MKs (p=0.0062[het], p=0.052[hom]). Accelerated megakaryopoiesis in CALRins5‑mutated clones was corroborated by upregulation of the late MK maturation marker NFE2 (p=0.0061[het], p=0.052[hom]), and an increased granularity of hom CALRins5 mutated vs. unmutated MKs (p=0.0089).
Conclusion
We demonstrate that het and hom CALRins5‑ and del52‑mutated iPS cells recapitulate features observed in ET and PMF patients, such as an increased number of MKs, and that this can be reversed by repair of the mutation, suggesting that mutant CALR is responsible for these effects. Mechanistically, our data show that mutant CALR induces an accelerated maturation process of MKs, which is in line with accelerated platelet turnover seen in ET and PMF patients.
Keyword(s): Megakaryocyte differentiation, Myeloproliferative disorder
Abstract: S196
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
In myeloproliferative neoplasms (MPN), somatic driver mutations of calreticulin (CALR), present in up to 30% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), include 52bp deletions (del52) and 5bp insertions (ins5) in exon 9. Mutant CALR acts by firmly binding to the thrombopoietin receptor (MPL), leading to constitutive activation of Janus kinases (e.g. JAK2) and associated downstream signaling. This results in cellular transformation, including enhanced megakaryopoiesis. The mechanism of increased megakaryopoiesis and the role of mutant CALR zygosity has not fully been elucidated to date.
Aims
Establishment of an MPN patient‑derived induced pluripotent stem (iPS) cell model focusing on CALR mutations in megakaryocytic development.
Methods
iPS cells were reprogrammed from PBMCs of MPN patients with CALRdel52 or CALRins5 mutations, or from healthy donors. Since reprogramming of CALR-mutant cells only resulted in homozygous (hom) and heterozygous (het) clones but not CALRwt clones, CALR mutations were repaired using CRISPR/Cas9n gene editing. iPS cells were differentiated into myeloid cells, with a particular focus on megakaryocytes (MKs). Bulk populations were analyzed by flow cytometry, and CD61+ MKs were purified and further analyzed for RNA expression and ultrastructural morphology.
Results
We established an iPS cell model including CALRins5 and del52 hom and het clones and CRISPR-repaired isogenic (WTcr) clones. Homozygous CALRdel52 and CALRins5 iPS clones were found to lack myeloperoxidase (MPO) activity, as has been described for patients with hom CALR mutations, and MPO activity as an indicator of CALR wt protein function was restored in WTcr clones. We established a protocol allowing efficient generation of iPS cell‑derived MKs with characteristics of primary MKs, such as formation of a demarcation membrane system, cytoplasmic pro-platelet protrusions, and MK-typical granules. We demonstrate that hom and het CALRins5- and del52-mutated iPS cells generate a significantly higher number of MKs than related WTcr clones (p<0.05 each). Moreover, MK generation of CALRins5 and del52 cells is TPO-independent in contrast to WTcr and healthy donor clones. Enhanced megakaryopoiesis was due to an accelerated maturation process of CALR‑mutated MKs, since the number of CD42b+ cells among the population of CD61+CD41+ MKs on day 10 was higher in hom (p=0.001[del52], p=0.18[ins5]) and het (p=0.044[ins5]) CALR‑mutated cells vs. WTcr MKs. In contrast, on day 14, the number of CD42+ MKs in CD61+CD41+ population was similar for all genotypes, suggesting that the WTcr MKs had taken longer to mature. Nonetheless, differences in the expression of megakaryocytic genes were observed between genotypes, with an increased MPL expression in CALRins5 MKs compared to WTcr MKs (p=0.0062[het], p=0.052[hom]). Accelerated megakaryopoiesis in CALRins5‑mutated clones was corroborated by upregulation of the late MK maturation marker NFE2 (p=0.0061[het], p=0.052[hom]), and an increased granularity of hom CALRins5 mutated vs. unmutated MKs (p=0.0089).
Conclusion
We demonstrate that het and hom CALRins5‑ and del52‑mutated iPS cells recapitulate features observed in ET and PMF patients, such as an increased number of MKs, and that this can be reversed by repair of the mutation, suggesting that mutant CALR is responsible for these effects. Mechanistically, our data show that mutant CALR induces an accelerated maturation process of MKs, which is in line with accelerated platelet turnover seen in ET and PMF patients.
Keyword(s): Megakaryocyte differentiation, Myeloproliferative disorder