P984: differential impacts of distinct mutation subtypes on altered s100a8 expression and phenotypic heterogeneity in calr-mutant mpn

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Numerous pathogenic CALR exon 9 mutations have been identified in myeloproliferative neoplasms (MPN), with type 1 (52bp deletion; CALRDEL) and type 2 (5bp insertion; CALRINS) being most prevalent. Despite the universal pathobiology of MPN driven by various CALR mutants, it is unclear why different CALR mutations result in diverse clinical phenotypes. Aims: To solve the enigma of disparate phenotypes in CALR-mutated MPN, we aim to explored through RNA sequencing (RNA-Seq) to dissect the transcriptomic dissimilarities between type 1 and type 2 CALR-mutated MPN. Methods:CALR-mutated MPN cells (CALRDEL, CALRINS and CALRWT control) were established through co-transduction of a human MPL-expressing lentiviral vector and an MSCV vector (constructed with either wild-type CALR, type 1 CALR, or type 2 CALR mutations) into BaF3 cells. RNA-Seq was employed to compare their transcriptomic profiling. Differentially expressed candidate genes were selected and subjected to in vitro analysis. Clinical samples from MPN patients were used for validation. Comparisons on phenotypic discrepancies were also made. Results: On RNA-Seq, we found that S100a8 was specifically enriched in CALRDEL but not in CALRINS MPN cells. Quantitative RT-PCR further confirmed the differential expression pattern of S100a8 between CALRDEL and CALRINS cells, whereas increased S100A8 protein was also exclusively seen in the cell culture supernatant of CALRDEL cells. With constitutive Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) being a hallmark phenomenon in MPN, we assessed through this pathway and revealed that CALRDEL cells treated with an STAT3 inhibitor would exhibit reduced S100A8 expression. Luciferase reporter assay targeting S100a8 promotor region which covered several putative STAT3-binding sequences confirmed the presence of contrasting transcriptional regulation between CALRDEL and CALRINS cells, whereas complementary STAT3 inhibition experiments attenuated the reporter activity in CALRDEL cells. Importantly, pyrosequencing demonstrated the presence of relative hypomethylation in two CpG sites within the potential pSTAT3-targeting S100a8 promoter region in CALRDEL cells as compared to both CALRWT and CALRINS cells, a result suggesting that distinct epigenetic alteration could factor into the divergent S100A8 levels in these cells. On functional analysis, knockdown of S100a8 with short hairpin RNA in CALRDEL cells resulted in decreased cellular proliferation with enhanced apoptotic activity. In clinical samples, we found the PB granulocytes from CALRDEL-mutated MPN patients exhibited enhanced S100A8 expression, a finding not observed in CALRINS-mutated patients. Finally, when segregating CALR-mutated MPN patients into S100A8High and S100A8Low subgroups, we consistently observed that those harboring higher S100A8 expression did have less escalated platelet counts, alluding to an intriguing, yet-to-be confirmed hypothesis that differential S100A8 activity might contribute to disparate thrombopoietic potential between CALRDEL and CALRINS MPN. Summary/Conclusion: Our work, through RNA-Seq followed by in vitro and clinical validations, provides an innovative perspective on how S100A8 may be differentially activated in type 1 CALR-mutated MPN, a finding that could offer potential implications for therapeutic and prognostic purposes. Keywords: Myeloproliferative disorder, Gene mutation, Phenotype, Promoter methylation