Myelodysplastic Syndromes – Basic and Translational Studies
Oral and Poster Abstracts
636. Myelodysplastic Syndromes – Basic and Translational Studies: Poster I
Hall A, Level 2
(Orange County Convention Center)
Silvia Buonamici, PhD1, Rachel Darman, PhD1*, Samantha Perino, BS1*, Anant Agrawal, PhD1*, Shouyong Peng, PhD1*, Michael Seiler, PhD1*, Kian Huat Lim, PhD1*, Erica B. Bhavsar2*, Jacob Feala, PhD1*, Esther A. Obeng, MD, PhD3, Suzanna Bailey, PhD1*, Betty Chan, PhD1*, Peter Fekkes, PhD1*, Gregg F. Keaney, PhD1*, Pavan Kumar, PhD1*, Kaiko Kunii1*, Linda Lee, MS1*, Crystal Mackenzie4*, Mark Matijevic4*, Yoshiharu Mizui, PhD1*, Khin Myint4*, Eunice Park1*, Ermira Pazolli1*, Michael Thomas1*, John Wang1*, Markus Warmuth, MD1*, Lihua Yu, PhD1*, Ping Zhu1*, Richard R. Furman, MD5, Benjamin L Ebert, MD, PhD6 and Peter G Smith, PhD1*
1H3 Biomedicine, Cambridge, MA
2Department of Hematology/Oncology, Weill Cornell Medical College, New York, NY
3Pediatric Hematology/Oncology, Boston Children's Hospital, Boston, MA
4Eisai, Andover, MA
5New York-Presbyterian/Weill Cornell Medical Center, New York, NY
6Division of Hematology, Brigham and Women's Hospital, Boston, MA
Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3’ splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3’ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS.
Disclosures: Buonamici: H3 Biomedicine:
Employment
. Darman: H3 Biomedicine:
Employment
. Perino: H3 Biomedicine:
Employment
. Agrawal: H3 Biomedicine:
Employment
. Peng: H3 Biomedicine:
Employment
. Seiler: H3 Biomedicine:
Employment
. Feala: H3 Biomedicine:
Employment
. Bailey: H3 Biomedicine:
Employment
. Chan: H3 Biomedicine:
Employment
. Fekkes: H3 Biomedicine:
Employment
. Keaney: H3 Biomedicine:
Employment
. Kumar: H3 Biomedicine:
Employment
. Kunii: H3 Biomedicine:
Employment
. Lee: H3 Biomedicine:
Employment
. Mackenzie: Eisai:
Employment
. Matijevic: Eisai:
Employment
. Mizui: H3 Biomedicine:
Employment
. Myint: Eisai:
Employment
. Park: H3 Biomedicine:
Employment
. Pazolli: H3 Biomedicine:
Employment
. Thomas: H3 Biomedicine:
Employment
. Wang: H3 Biomedicine:
Employment
. Warmuth: H3 Biomedicine:
Employment
. Yu: H3 Biomedicine:
Employment
. Zhu: H3 Biomedicine:
Employment
. Furman: Acerta Pharma BV:
Research Funding
; Gilead:
Consultancy
; Pharmacyclics LLC, an AbbVie Company:
Consultancy
,
Honoraria
,
Speakers Bureau
. Ebert: Celgene:
Consultancy
; H3 Biomedicine:
Consultancy
; Genoptix:
Consultancy
,
Patents & Royalties
. Smith: H3 Biomedicine:
Employment
.
*signifies non-member of ASH