P1008: the telomerase inhibitor imetelstat differentially targets jak2v617f- versus calr-mutant myeloproliferative neoplasm cells and inhibits jak-stat signaling

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Imetelstat, a telomerase inhibitor, has shown clinical activity in patients with Myeloproliferative Neoplasms (MPN), including primary myelofibrosis (PMF) and essential thrombocythemia (ET). Imetelstat is an oligonucleotide with a nucleotide sequence that is complementary to and therefore specifically binds with high affinity to the template region of the RNA component of human telomerase and acts as a potent, competitive inhibitor of telomerase enzymatic activity. Although inhibition of telomerase by imetelstat leads to telomere length (TL) shortening, its mechanisms to induce responses in MPN need further elucidation. Aims: To analyze clone evolution of the MPN mutation profile during a two-year course of imetelstat treatment through disease progression; to assess for mechanistic differences in MPN cells harboring JAK2V617F versus CALRdel52 mutations. Methods: A high-risk PMF patient with multiple disease-related mutations who had been heavily pre-treated and was without disease control while on a combination of ruxolitinib and hydroxyurea, achieved a good response initially, when being treated with imetelstat within the MYF2001 clinical trial. TL was measured via flow-FISH during 30 months of treatment. Induced pluripotent stem cells (iPSC) were generated, carrying the dominant mutations during disease progression (JAK2V617F, ASXL1T880fs, KRAST58I, TET2E1470fs and U2AF1Q157R). A “spin EB” (embryoid bodies) protocol was used for hematopoietic differentiation of the established iPSC clone, and the cells were subsequently treated with imetelstat in vitro. In addition, imetelstat response was analyzed in human TF-1MPL and murine 32DMPL cells stably expressing JAK2V617F or CALRdel52. Results: We confirmed continuous shortening of TL (8.09 kb to 5.49 kb in granulocytes after 21 months) but observed an increase of a KRAST58I mutated clone (2.53% to 52% allele burden in 2 years of treatment) with additional JAK2V617F, ASXL1T880fs, TET2E1470fs and U2AF1Q157R mutations during the patient’s clinical course on imetelstat. We successfully generated iPSC with the complex mutational profile found in the patient´s blood cells. The iPSC were differentiated to hematopoietic stem and progenitor cells (HSPC) and subsequently to myeloid cells. We demonstrated that imetelstat reduced clonogenic growth of the patient’s primary CD34+ cells (p=0.0206) but not of the iPSC-derived CD34+ cells. iPSC-derived myeloid cell differentiation to megakaryocytes and granulocytes was not hampered. However, TL of the iPSC-derived HSPC, which was overall increased during the reprogramming process (mean: 13.9 kb), was reduced upon imetelstat treatment for 14 days (loss of 14.02% ± 5.42; p=0.011). Next, using the human TF-1MPL and murine 32DMPL cell lines, we demonstrate a stronger effect of imetelstat on CALRdel52-positive vs. JAK2V617F-positive cell viability (p=0.0361 and p=0.0311 for 5 µM imetelstat, respectively), and this was associated with an immediate downregulation of JAK2 protein phosphorylation and downstream signaling as well as a reduction of telomerase reverse transcriptase (hTERT) and STAT3 mRNA expression. Summary/Conclusion: Our data demonstrate that imetelstat reduces hTERT expression and TL, and targets JAK/STAT signaling, particularly in CALR-mutated cells. Although the exact patient population who will benefit most from imetelstat is still undefined, our data propose that CALR-mutated clones are highly vulnerable. Keywords: Mutation analysis, Myeloproliferative disorder, Myeloid differentiation, Telomere length