The Activated TGFβ Pathway in Shwachman Diamond Syndrome Impairs Hematopoiesis and Is Down-Regulated By Deletion of 7q
Authors: Ruiz-Gutierrez Melisa , Joyce Cailin , Vargel Bolukbasi Ozge , Kotini Andriana , Alexe Gabriela , Whangbo Jennifer , Russell David , Stegmaier Kimberly , Novina Carl D. , Papapetrou Eirini P. , Shimamura Akiko 
Published: April 2021

Abstract
Monosomy 7 or deletion of 7q (del(7q)) are common cytogenetic abnormalities in pediatric MDS. Monosomy 7/del(7q) frequently arises in the context of inherited bone marrow failure (BMF) syndromes such as Shwachman Diamond Syndrome (SDS), an autosomal recessive disorder caused by biallelic mutations in the SBDS gene. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited BMF. The basis for this propensity to develop monosomy 7/del(7q) remains unclear. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in BMF, remains a critical question. The aim of this study is to investigate the molecular consequences of del(7q) in the context of BMF with the goal of developing more effective treatments.
The lack of synteny between murine and human chromosome 7 has posed a major barrier to modeling monosomy 7/del(7q). Therefore, we utilized SDS patient-derived induced pluripotent stem cells (SDS-iPSC), which recapitulate the hematopoietic defects of this BMF syndrome. Independent SDS-iPSC lines were derived from two patients with SDS. Proliferationof SDS-iPSCs was reduced relative to wild type controls without a concomitant increase in cell death. Compared to normal iPSC controls, SDS iPSC-derived CD34+ cells exhibited reduced hematopoietic differentiation to CD45+ cells, decreased erythroid and myeloid hematopoietic progenitor colony formation in methylcellulose, and impaired terminal myeloid differentiation.
To study the molecular and biologic effects of del(7q), a deletion of the MDS-associated region of the long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q in SDS iPSCs spanned band q11.23 to q36.3. The SDS-del(7q) iPSCs expressed stem cell markers and formed teratomas as efficiently as their isogenic SDS iPSC controls.Deletion of 7q failed to confer a relative growth advantage even within the context of BMF. Indeed, proliferation of the SDS-del(7q) iPSCs was reduced below that of both the isogenic SDS iPSCs and normal controls without an increase in cell death. To investigate the effect of del(7q) on hematopoiesis, iPSC-derived CD34+ cells were assayed for their ability to undergo hematopoietic differentiation. SDS-del(7q) CD34+ cells demonstrated reduced differentiation to CD45+ cells, reduced methylcellulose hematopoietic progenitor colony formation and impaired terminal myeloid differentiation compared with isogenic SDS CD34+ cells. These data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic SDS iPSCs and results in a further impairment of hematopoiesis.
To gain insight into the mechanisms of del7q-associated clonal evolution in SDS, we performed RNA sequencing (RNAseq) of SDS and SDS-del(7q) iPSC. Expression of TGFβ pathways and their downstream targets were reduced in SDS del(7q) iPSCs compared to isogenic SDS iPSC controls. This observation was intriguing because single cell RNAseq analysis of primary SDS bone marrow cells revealed that TGFβ pathway is hyperactivated in SDS. Additionally, a quantitative DNA aptamer-based proteomic analysis demonstrated elevated levels of TGFβ ligands in plasma from six SDS patients compared to 6 age-matched controls. Western blot analysis showed increased P-SMAD2 levels in SDS iPSCs compared to SDS-del(7q) and normal controls, while total levels of SMAD2 were unchanged. Pharmacological targeting of TFGβ with small molecule inhibitors resulted in selective improvement of SDS hematopoietic colony formation without stimulating outgrowth of the isogenic SDS-del(7q) cells. Although del(7q) does not seem to confer an advantage in growth or hematopoiesis in vitro, we are testing whether the deletion of 7q might confer relative resistance to elevated TGFβ levels in vivo, resulting in an overall survival advantage of the del7q clone.These results demonstrate that the TGFβ pathway is activated in SDS, and that deletion of 7q reverses the TGFβ pathway hyperactivation in SDS. Inhibition of TGFβ selectively rescues hematopoiesis in SDS but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth.

Disclosures
Stegmaier: Novartis: Consultancy, Research Funding. Shimamura: TransCellular Therapeutics: Other: Spouse is majority shareholder.