Oncogenic Mutations in XPO1 Promote Lymphoid Transformation By Altering Nuclear/Cytoplasmic Localization of NFκB Signaling Intermediates

Genomic analyses across cancer have identified recurrent somatic mutations in XPO1, which encodes a major nuclear cytoplasmic transport protein responsible for exporting a broad range of cargo proteins from the nucleus to the cytoplasm. Specifically, heterozygous mutations at the XPO1 hotspot residue E571 (most frequently E571K) are recurrent in B-cell malignancies including chronic lymphocytic leukemia, primary mediastinal B-cell lymphoma, and Hodgkin lymphoma. Despite the fact that XPO1 is the target of numerous small molecules in clinical development, functional evidence for precisely how altered XPO1 expression or mutations might promote transformation is not well delineated.

We utilized genome editing to generate isogenic B-cell leukemia cells for physiologic expression of the XPO1 E571K mutation from the endogenous XPO1 locus. The heterozygous XPO1 E571K mutant cells had increased cell proliferation in vitro and in vivo relative to XPO1 wild-type (WT) counterparts. The effects of XPO1 mutation were distinct from XPO1 genetic depletion, which completely suppressed cell growth (Figure A). Given the high conservation of XPO1 across mammals, we next generated a conditional knock-in mouse model to express the Xpo1 E571K mutation from the endogenous Xpo1 locus. Cd19- Cre Xpo1 E571K/WTmice were born at normal Mendelian ratios, but were smaller than littermate controls and had increased number of B-cells in the blood and transitional (T1, T2) B-cells in the spleen with splenomegaly (Figure B). Colony forming assays in IL-7-containing methylcellulose using bone marrow (BM) from these mice revealed increased number of B-cell progenitors with enhanced clonogenicity.

Given several recent reports suggesting that XPO1 mutations are clonal early events in lymphomagenesis, we also evaluated the effects of pan-hematopoietic expression of Xpo1 E571K. Transplantation of CAG- CreERT Xpo1 E571K/WTBM cells followed by tamoxifen administration to recipients resulted in skewing to B-lymphopoiesis and thrombocytopenia 12 weeks after tamoxifen induction. BM cells from CAG- CreERT Xpo1 E571K/WTshowed strikingly increased B-cell colony formation and serially replating capacity in IL-7 with reduced clonogenicity in myeloid cytokine-containing methylcellulose (Figure C). We next crossed the Cd19- Cre Xpo1 E571K/WTmice to transgenic mice with pan-hematopoietic BCL2 overexpression (vav-BCL2 mice). By 8 weeks, Cd19- Cre Xpo1 E571K/WT/ vav- BCL2 mice had higher B-cell lymphocytosis, increased B-cells in the BM and splenomegaly compared to Cd19- Cre Xpo1 WT/WT, Cd19- Cre Xpo1 E571K/WTor Cd19- Cre / vav - BCL2mice, suggesting cooperativity between the proliferative effects of XPO1 E571K and anti-apoptotic effects of BCL2 in B-cells. Moreover, B-cell progenitors from Cd19- Cre Xpo1 E571K/WT/ vav-BCL2 mice again demonstrated remarkable clonogenic capacity in IL-7-containing media compared with other genotypes.

To understand the mechanism by which the XPO1 hotspot mutation confers growth-promoting effects, we performed mass spectrometry on fractionated nuclear and cytoplasmic lysates from XPO1 E571K knock-in and parental cell lines (Figure D). Numerous members of the K63-ubiquitination, TLR4, and NFκB pathways were differentially exported in XPO1 E571K mutant cells. Given the known oncogenic role of NFκB signaling in lymphoid malignancies, we further functionally evaluated this pathway. XPO1 mutant cells demonstrated differential localization of cIAP1/2, NFkBIB, IKKB, TAB2 and TRAF2. Differential export of NFκB signaling intermediates was confirmed by western blot and immunofluorescence revealing increased (but not complete) retention of cIAP1/2 proteins in the nucleus and increased levels of TRAF2 in the cytoplasm of mutant cells compared to WT. Phenotypically, XPO1 E571K cells demonstrated higher baseline NFkB signaling and transcriptional responses to stimulation with TNF-α than XPO1 WT counterpart cells (Figure E).

Overall, these data identify that hotspot mutations in XPO1 promote lymphoid biased hematopoiesis and transformation by altering nuclear export of key proteins involved in hematopoietic regulation. Although numerous cellular pathways are altered in XPO1 E571K mutant cells, this mutation prominently affects compartmentalization of NFκB signaling components in a manner that promotes activation of this pathway and contributes to lymphomagenesis.

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