XBP1 PROMOTES PRE-B CELL ACUTE LYMPHOBLASTIC LEUKEMIA THROUGH OF IL-7 RECEPTOR SIGNALING IN MICE
Author(s): ,
Azam Salimi
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
,
Margherita Vieri
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
,
Mirle Schemionek
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
,
Michael Huber
Affiliations:
Institute of Biochemistry and Molecular Immunology,Medical Faculty, RWTH Aachen University,Aachen,Germany
,
Tim Henrik Brümmendorf
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
,
Behzad Kharabi Masouleh
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
Iris Appelmann
Affiliations:
Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,Medical Faculty, RWTH Aachen University,Aachen,Germany
EHA Library. Salimi A. Jun 14, 2019; 265942; PF152
Azam Salimi
Azam Salimi
Contributions
Abstract

Abstract: PF152

Type: Poster Pitch

Presentation during EHA24: On Friday, June 14, 2019 from 17:30 - 19:00

Location: Poster area

Background

Activating RAS mutations such as NRASG12D drive one third of pre-B cell acute lymphoblastic leukemia (pre-B ALL). RAS mutations are a major cause of resistance to conventional chemotherapy or targeted therapy like tyrosine kinase inhibitors (TKIs). In addition, targeting of leukemia stem cells (LSCs) as the fundamental source of relapse remains challenging with  currently available therapies.

Recently published studies identified the unfolded protein response (UPR) as critical for pre-B ALL survival and high expression of XBP1 correlated with a poor prognosis of ALL patients. In addition, clinical data demonstrated that the UPR genes are up-regulated in pre-B ALL. However, the mechanism of dependency on the UPR-dependency and particularly its IRE-1α-XBP1 axis is not yet elucidated in RAS mutated Pre-B ALL.

Aims

In this study we aimed to identify the molecular signature of IRE-1α-XBP1 signaling at the different stages of RAS-mediated leukemogenesis. Subsequently, we aimed to decipher the mechanism behind of UPR-dependency of RAS mutated pre-B ALL.

Methods

We employed a TET-ON inducible NRASG12D model in conditional Xbp1 knockout mice. For this purpose, we transduced IL-7-dependent murine Xbp1fl/fl pre-B cells with a TET-ON inducible NRASG12D retrovirus. Thereafter the TET-ON NRASG12D Xbp1fl/fl pre-B ALL cells are transduced with inducible-Cre. We performed in vitro cell cycle and apoptotic assays using propidium iodide (PI) and Annexin-V / 7-AAD staining, respectively. Furthermore, Western Blot and qRT-PCR were performed to analyze target gene expression. In a second approach to assess of the efficacy of the IRE-1α inhibitor MKC-8866, we focused on the signaling events following pharmacological inhibition of XBP1 activation.

Results

To better understand the role of Xbp1 at the different stages of progression of pre-B ALL, we examined Xbp1 transcript levels in three different cell types: normal pre-B cells, early pre-B ALL cells and established pre-B ALL. We demonstrated that Xbp1 expression increased upon activation of NRASG12D. To determine the significance of Xbp1 in pre-B ALL, we genetically deleted the IRE-1α target Xbp1 using Cre-mediated deletion of Xbp1fl/fl in our mouse model of pre-B ALL. Genetic loss of Xbp1 strongly induced apoptosis (2.4-fold, p<0.0001) and caused cell cycle arrest (induction of G0/1, 1.9-fold, p=0.0001). In addition, genetic deletion of Xbp1 function decreased p-STAT5-pY694 and slightly increased pro-apoptotic BIM protein and cell cycle negative regulator P21cip1 (1.7-fold, p<0.0001). Pre-B ALL cells in the absence of active XBP1 also increased RAS downstream targets p-ERK-T202/Y204 and MAPK negative regulators such as Dusp6 (1.4-fold p=0.0004) but decreased expression of Dusp10 (1.6-fold, p=0.0011). Interestingly, pharmacological inhibition of Xbp1 activation using MKC-8866 or incubation of pre-B ALL cells without IL-7 resulted in similar effects on target genes expression in comparison to the genetic deletion of Xbp1.

Conclusion

In summary, our work strongly supports the hypothesis that XBP1 is critical for progression and maintenance of RAS mutated leukemogenesis. Reduction of active transcript of Xbp1 by MKC-8866 or genetic loss of Xbp1 did reduce STAT5 as a downstream linchpin of the IL7 receptor signaling pathway and resulted in increased ERK1/2 as a downstream target of RAS pathway. Xbp1 expression is positively regulated by STAT5. Furthermore, loss of XBP1 might induce negative feedback on upstream target STAT5 and inhibit proliferation and induce apoptosis via the ERK signaling pathway.

Session topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research

Keyword(s): Acute lymphoblastic leukemia, IL-7, Ras, STAT5

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