Interplay between PI3K/mTOR Signaling and IRE1a-XBP1 Promotes Survival of Pre-B NRAS^G12D all Cells Providing a Therapeutic Vulnerability for the "Undruggable" Driver RAS

Background

Activating RAS mutations drive around 30% of pre-B cell acute lymphoblastic leukemias (pre-B ALL) and are particularly common in relapsed ALL with a consecutively poor outcome. Recently published data demonstrated the critical role of the unfolded protein response (UPR) network, namely its IRE1α-XBP1 axis, for the survival of pre-B ALL cells: High expression of XBP1 confers a poor prognosis in pre B-ALL. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. In this study, we aimed at identifying the molecular mechanism underlying pro-survival IRE1α-XBP1 signaling in RAS mutated pre-B ALL.

Methods

For a TET-ON inducible NRAS^G12D model in conditional Xbp1 knockout mice, we used interleukin 7 (IL-7)-dependent murine Mx1-Cre;Xbp1^fl/fl pre-B cells transduced with a TET-ON inducible NRAS^G12D. We performed in vitro cell cycle and apoptosis assays with propidium iodide (PI) and annexin-V/PI. Furthermore, Western Blot and RT-qPCR were applied to analyze target gene expression. In a second approach, we focused on the signaling events following the blockade of RAS downstream targets using the MEK inhibitor PD0325901 and the dual PI3K/mTOR inhibitor BEZ235. We then assessed the efficacy of small molecule inhibition of IRE1α by MKC-8866 on XBP1 inactivation in RAS-mutated pre-B ALL cells either as a single treatment and in combination with the above mentioned drugs.

Results

We found the expression of Xbp1 significantly increased at the mRNA level with induction of NRAS^G12D. To determine the significance of Xbp1 in NRAS^G12D-driven pre-B ALL, we genetically deleted the IRE1α target Xbp1 using Cre-mediated deletion of Xbp1^fl/fl in our mouse model of pre-B ALL. Genetic loss of Xbp1 significantly induced apoptosis (2.0-fold, p<0.0001) and caused cell cycle arrest (induction of G_0/1, 1.7-fold, p=0.0003) along with an increase in the expression of CDK inhibitors, p21^CIP1 and p27^KIP1 at the protein level. Genetic ablation of Xbp1 abrogated IL-7 receptor (IL-7R) signaling by reducing the phosphorylation levels of STAT5-Y⁶⁹⁴ and JAK1-Y¹⁰²²/Y¹⁰²³. In an additional approach, we revealed that IL-7-deprived pre-B ALL cells reduce the mRNA expression of Xbp1s, indicating that Xbp1 acts as a downstream linchpin of the IL-7 receptor signaling pathway.

Both IL-7-deprivation and genetic loss of Xbp1 increased the phosphorylation levels of ERK1/2-T²⁰²/Y²⁰⁴, AKT-S⁴⁷³ and the protein levels of NRAS^G12D and MAPK negative regulator DUSP6. Pharmacological inhibition of XBP1 activation using MKC-8866 resulted in similar effects on the expression of RAS downstream targets. We therefore tested MKC-8866 in combination with MEK inhibition by PD0325901 as a potential therapeutic strategy against pre-B ALL, which proved non-efficient. As a second option with therapeutic implications, we focused on the PI3K pathway which acts downstream of both the IL-7R and RAS signaling pathways. Strikingly, we observed that genetic ablation of Xbp1 (viable cells after 72 h, BEZ: 71.9 ± 9.0 vs BEZ+ Mx1-Cre;Xbp1^fl/f: 10.0 ± 4.9) or pharmacological inhibition of its production with MKC-8866 (viable cells after five days, BEZ: 58.0 ± 6.8 vs BEZ+ MKC-8866: 13.3 ± 7.4) sensitizes pre-B ALL to dual inhibition of PI3K/mTOR with BEZ235. By applying the Bliss formula, we were able to show that BEZ235 in combination with MKC-8866 synergistically reduces the viability of RAS-mutated pre-B ALL cells. Gene expression analysis indicated that BEZ235 in combination with MKC-8866 fully blocked IL-7R signaling and caused an aberrant activation of Ras-Erk signaling. Targeting PI3K/mTOR signaling along with XBP1 inactivation increased expression of NRAS^G12D and its target DUSP6. In addition, we showed that combined therapy increased expression levels of p19^Arf in RAS-mutated pre-B ALL, implicating cell senescence mediated by activated RAS signaling.

Conclusion

Our work strongly supports the hypothesis that XBP1 induces its positive effects on progression of pre-B ALL cells through the IL-7R signaling pathway. IL-7R signaling through its downstream effector XBP1 counteracts the RAS signaling pathway to promote leukemogenesis in pre-B ALL cells. Active XBP1 prevents the cytotoxic effects of BEZ235 in pre-B ALL cells, and hence targeting XBP1 in combination with dual PI3K/mTOR inhibition by BEZ235 appears as a promising targeted strategy against the "undruggable" driver RAS in NRAS^G12D-mutated pre-B ALL.