MSI2 and TGFβR1 Expression Levels in CML Patients and Hematopoietic Cell Lines

Musashi 2 (also known as MSI2), a mRNA binding protein is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting translation. MSI2 is preferentially expressed in hematopoietic tissue, in particular early myeloid progenitors. Moreover, investigators suggest upregulated MSI2 disrupts regulatory pathway/s leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML (Kharas et al. Nat Med. 2010; 16:903; Ito et al. Nature. 2010; 466:765). Indeed we have confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in chronic phase (CP), irrespective of lymphoid or myeloid transformation. Furthermore, we have shown MSI2 and BCR-ABL1 expressions correlate. Here we report data implying MSI2 functions viaTGFβ1 signalling pathway.

We retrospectively studied 54 CML cDNA samples from 34 patients (M:15 ; F:19) in CP with median age 55.5 years (range 12-74). Apart from 3 patients treated with interferon+AraC, the remainder were prescribed Imatinib mesylate. Of the 54 samples 29 were collected at diagnosis (Dx) and 25 were obtained at 3 months post therapy (3M). For 20 of the patients, samples collected at Dx and 3M were available. Eight of the patients failed to achieve major molecular response (MMR) within 12 months post therapy. In addition to patient samples we included 19 normal cDNA controls from healthy blood donors (M:10 ; F:9), with 41 years median age (range 20-61). We also included 20 cDNA samples derived from hematopoietic cell lines (lymphoma: 7; AML: 3; CML:6 ; essential thrombocythemia: 1 ; hyperesoinophilic syndrome: 1 ; Acute lymphoblastic leukemia: 1). The cDNA was synthesized using reverse transcriptase and random hexamers. All the samples were subjected to quantitative real time PCR using TaqMan assay to quantify MSI2, TGFRβ1 and GUSβmRNA levels. Furthermore, we subjected protein isolated from the cells lines to Western blot analysis to assess TGFβR1 expression.

MSI2 mRNA median levels in patient samples were significantly decreased compared with the NC group at Dx and at 3M, p=0.002 and p=0.013, respectively. But we found no significant difference in MSI2 mRNA levels at Dx nor at 3M, between those who achieved major molecular response and those who did not within 12 months of starting therapy, p=0.215 and p=1.871, respectively. Also we observed no significant difference between the patient samples and the NC group TGFβR1 transcript numbers at Dx nor at 3M, p=0.057 and p=0.097, respectively. Equally we observed no significant difference in TGFβR1 levels, either at Dx or 3M, between those who did and failed to achieve MMR. But we found strong to moderate correlation between MSI2 and TGFβR1 expression when we compared the Dx and 3M data as determined by Spearman correlation coefficient, r=0.6975 and 0.5715, respectively. More importantly, we observed 9 of the cell lines, with increased (>6.7%) or detectable MSI2 mRNA expression, of which 6 express BCR-ABL1 (BV173, Lama87, K562, KCL22, KU812 and SupB15), the TGFβR1 protein was considerably decreased. The BCR-ABL1 positive cell line Meg01 was the one exception with increased MSI2 mRNA levels (7.1%) and clearly detectable TGFβR1 protein. Conversely, of the 10 BCR-ABL1 negative cell lines with decreased or undetectable MSI2 transcripts the TGFβR1 protein was clearly detectable in 8. Of these 7 were lymphoid cell lines (L-248, KM-H2, L-1236, HDLM-2, BJAB, U-266, U-2932) and one was essential thrombocythemia, SET2. The 2 exceptions were AML cell lines OCI-AML-2 and OCI-AML-3 with considerably decreased MSI2mRNA levels and markedly decreased TGFβR1 protein expression.

We suggest MSI2 expression is significantly decreased in CML patients in CP because the early progenitors expressing it are masked by large bulk of mature myeloid cells in these patients. Therefore to assess the prognostic value of MSI2 levels at diagnosis it should be quantified in CD34+ enriched samples at diagnosis. More importantly, finding cell lines expressing MSI2, with the exception of 3 cell lines (OCI-AML-2, OCI-AML-3 and Meg01) had decreased TGFβR1 protein is consistent with its role as a post transcription regulator. These findings combined with the previous data showing MSI2 mRNA levels are increased in CML BC support the notion that it functions via TGFβR1 signaling pathway to influence CML transformation.