Hematopoietic Cell Lines and Patient Samples Show a Correlation Between Upregulated MSI2 and BCR-ABL Expression
Authors: Kaeda Jaspal S. , Schwarzer Rolf , Slany Robert K. , Burmeister Thomas , Vagge Elisabetta , Amini Leila , Ringel Frauke , Pardon Katarina , Schwarz Michaela , Dörken Bernd , Jundt Franziska , le Coutre Philipp 
Published: October 2019

Abstract
Increased expression of Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to trigger progression of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC), which is frequently fatal. The data imply that MSI2 is upregulated by HOXA9 leading to disruption of the critical hematopoietic stem cell (HSC) fate decisions via the NUMB-NOTCH signaling pathway. These events lead to HSC proliferation, impaired myeloid differentiation and are associated with worse prognosis in CML and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). Previously, we confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in CP, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). To further elucidate this regulatory pathway we assessed whether HOXA9 expression correlated with increased MSI2mRNA levels and upregulation of NOTCH.
Because of the rarity of BC samples and finding MSI2 increased in lymphoid and myeloid BC CML patients, we studied lymphoid [n=12 (Lymphoma:8; Myeloma:3; ALL:1)] and myeloid [n=12 (AML:4; CML:6; HES:1; ET:1)] cells lines. We also included 29 BCR-ABL positive (e1a2: 18; e13a2: 6; e14a2: 5) diagnostic samples from acute lymphoblastic leukaemia (ALL) patients (M:18; F:11); median age 50 years (range: 26-74). We quantified MSI2 and HOXA9 mRNA transcripts by quantitative PCR and expressed the data as % ratio of the control gene, GUSβ.Western blotting of cell line protein extracts was performed to assess expression of NOTCH 1, 2, 3 receptors and delta like ligand 3.
We found MSI2 [median: 7.10% (range:0.06-38.71)] and HOXA9 [median: 2.40% (range:0.00-51.13)] expression was generally restricted to myeloid cell lines. Of the 12 lymphoid cell lines, MSI2 was only detectable in SUPB15 with 8.44%. The latter is a B-lymphocyte ALL cell line, known to express BCR-ABL e1a2 transcript. Similarly, HOXA9 [median: 0.00 (range:0.00-27.0%)] expression ranged from undetectable to 0.08%, among this group, apart from HD-MY-Z with 27.0%. In contrast, HOXA9 [median: 0.02% (range:0.01-2.18)] and MSI2 [median: 3.58% (range: 1.24-22.38)] transcripts were detectable in all 29 BCR-ABL positive ALL patients, irrespective of the transcript type expressed. Among the ALL samples 7 (24%) had increased MSI2 levels, i.e. >6.7% (Kaeda, et al. Blood. 2011;118;supplement) and of these 6 expressed e1a2 transcript and the other e13a2. In summary, upregulated MSI2 expression was observed in 17 (32.0%) of the 53 samples screened. But only 4 [KG1, EOL1, HEL and MEG-01 (all myeloid cell lines)] of the 17 also had increased HOXA9 levels. Remarkably, 5 (62%) of the 8 myeloid cell lines with increased MSI2 are known to express BCR-ABL. NOTCH1 receptor was detectable in all the lymphoid cell lines. But, NOTCH expression was highly variable in myeloid cell lines. Overall, an upregulated MSI2 mRNA expression was not reflected in the NOTCH receptor levels nor in the HOXA9levels.
Our observations are consistent with MSI2 being limited to myeloid linage. However, in contrast to earlier reports our data imply that MSI2 functions via a pathway other than NOTCH signaling and is not regulated by HOXA9 alone. But the cell lines and ALL patients’ data provide further evidence of correlation between MSI2 and BCR-ABL expression, suggesting they interact, directly or indirectly, to arrest cell differentiation and trigger BC. These findings, together with our reported data, show increased MSI2 levels to be an important biomarker of poor prognosis and are likely to have an impact in optimizing clinical management. It also represents a potential novel therapeutic target, especially for the BCR-ABL positive stem cells resistant to tyrosine kinase inhibitors.

Disclosures:
Kaeda: Novartis: Research Funding. le Coutre:Novartis: Research Funding.