Interferon Alpha Activates the Unfolded Protein Response Resulting in Synergy with Bortezomib in JAK2 V617F Myeloproliferative Neoplasms

Introduction

Whilst rare, the transformation to accelerated or blast phase of a myeloproliferative neoplasm (AP/BP-MPN) is devastating. Patients rarely present with chemo-sensitive disease, and median survival rates in those treated with intensive or non-intensive regimens are limited to only a few months. New tolerable treatment approaches that target and eradicate the mutant clone are therefore urgently required. Interferon alpha (IFN-α) is a frontline therapy in chronic phase MPN and can induce sustained molecular responses in JAK2 V617F positive patients through targeted effects on the mutant clonal population. Unfortunately, these responses are modest in most cases and are gradual, taking many years to develop. Identification of new combination treatments which potentiate the effect of IFN-α in MPN cells may offer a new disease modifying approach to benefit patients with advanced and aggressive MPN phenotypes.

Aim

IFN-α was observed to induce apoptosis in JAK2 V617F positive cell line models. We set out to establish the mechanism of apoptosis induced in these cells to identify and exploit IFN-α induced therapeutic vulnerabilities with rational combination treatments.

Results

We developed an IFN-α resistant UKE1 MPN cell line (UKE1-NR) and directly compared it to the IFN-α responsive parental model (UKE1-P) by RNA-Seq. Following exogenous IFN-α therapy, both UKE1-P and UKE1-NR upregulated canonical IFN pathways and increased phosphorylation of STAT1 to a similar extent. Responsive UKE1-P cells demonstrated transcriptional upregulation of the PERK/ATF4/CHOP arm of the unfolded protein response (UPR) and the pro-apoptotic gene PMAIP1 (NOXA), which has previously been identified as a downstream target of this pathway. This was not observed in the UKE1-NR cells. Using qPCR, we validated this in JAK2 V617F positive UKE1, HEL and SET2 cells, observing upregulation of the UPR genes ATF3 and DDIT3 (CHOP) as well as PMAIP1 (NOXA) following IFN-α exposure.

Using ATAC-seq, we observed loss of chromatin accessibility in the UKE1-NR cells at genetic loci identified as binding sites for ATF3, ATF4 and CHOP, consistent with a functional role for these transcription factors. Small molecule inhibition of ATF4 activation using ISRIB antagonised the effect of IFN-α in responsive UKE1-P cells highlighting an important role for this pathway in mediating the response.

Bortezomib (Bz), a proteasome inhibitor, is a known inducer of the UPR. Combination of Bz with IFN-α synergistically increased apoptosis in all three JAK2 V617F cell line models. Furthermore, combination therapy resulted in significantly increased expression of ATF3, DDIT3 (CHOP) and PMAIP1 (NOXA) in comparison to single agent treatment. Importantly this combination was also determined to be synergistic in UKE1-NR cells despite non-response to single agent IFN-α. Moreover, the efficacy of this combination appears to be p53-independent as it was observed in TP53 wild type (UKE1) and mutant (HEL/SET2) cells. Additionally, the combination is effective following CRISPR knockdown of TP53 in HEL cells. CRISPR knockout of PMAIP1 (NOXA) subsequently demonstrated partial dependence on NOXA for the effectiveness of this combination. NOXA upregulation can sensitise to BCL2 inhibition, and in keeping with this, we have observed enhanced sensitivity to the clinically-relevant BCL2 inhibitor venetoclax and synergy in combination with Bz/IFN-α.

Conclusion

IFN-α can induce apoptosis in a range of JAK2 V617F positive cells. We have demonstrated that this apoptosis is characterised by a transcriptional upregulation of the UPR and NOXA and induces a therapeutic vulnerability which can be effectively exploited in combination therapy with bortezomib in a TP53 independent manner. These early pre-clinical results using readily available drug combinations offer a potential novel therapeutic approach in combination with or without BCL2 inhibition for patients with aggressive forms of MPN including AP/BP-MPN and will be taken forward for further study.