Elongator Complex Regulates MCL1 Dependency Via IRE1-XBP1 Axis of the ER Stress Response Pathway in Multiple Myeloma

Evasion of apoptosis is a hallmark of cancer wherein overexpression and amplification of pro-survival BCL2-family genes like MCL1 is a common observation. MCL1 is frequently amplified in many hematological cancers like Multiple Myeloma (MM) that depend on it for survival. BH3 mimetic drugs, like the BCL2-specific inhibitor Venetoclax, have been successfully used in the clinic to treat certain cancers, and MCL1-selective inhibitors are currently in clinical development. While inhibition of MCL1 displays promising preclinical activity, many cancer models display acquired or intrinsic resistance to MCL1 inhibitors (MCL1i). As MCL1-targeted therapies progress clinically, understanding mechanisms that lead to resistance will be important to not only identify therapeutically-exploitable targets to combat resistance, but to also determine if these biomarkers could stratify patients most likely to respond to an MCL1i.

Here, we used a genome-wide CRISPR knock-out screen to identify mechanisms of resistance to MCL1i AZD5991 in two MM cell lines, KMS11 and KMS34. We used a sgRNA library consisting of about 118,000 sgRNAs (~6 sgRNAs/gene), and treated the cells with DMSO or 1uM AZD5991 for 16 days (5 doublings). We identified 316 genes in KMS11 and 184 genes in KMS34 with >4-fold enrichment of sgRNAs; and 221 genes with >2-fold enrichment of sgRNAs in both cell lines. The sgRNAs targeting BAK and BAX were the most enriched overlapping hits. Using GSEA analysis of the 221 common genes with enriched sgRNAs, we discovered that the tRNA wobble uridine modification as the most enriched pathway. The tRNA U34 mcm5s2 modification is catalyzed by the elongator complex ELP1-6 and cytosolic thiouridylase CTU1/2.

Each subunit of the elongator complex is essential for its function and loss of any subunit results in destabilization of the complex. By knocking out ELP4 in five MM cell lines (KMS11, KMS34, KMS12-PE, MM.1S, and RPMI-8226), we first validated the destabilization of the complex by showing a robust decrease in the protein levels of ELP1 and ELP3 via western blot. As the elongator complex has additional functions, we also knocked-out tRNA U34 modification pathway specific CTU1 in KMS11, KMS34, and KMS12-PE cells. We showed that genetic knock-out of ELP4 and CTU1 results in increased resistance to MCL1i in all cell lines tested. We observed the highest increase in MCL1i resistance upon ELP4-KO in KMS11 and RPMI-8226 cell lines.

To understand the mechanism behind elongator complex mediated regulation of MCL1 dependency, we performed RNAseq and global proteomics in KMS11 cells (Parental, non-targeting control [NTC], ELP4-KO and CTU1-KO) and RPMI-8226 cells (Parental, NTC, and ELP4-KO). We show that the elongator complex is a regulator of IRE1-XBP1 axis of the ER stress response pathway; and knockout of IRE1 also results in MCL1i-resistance in KMS11 and RPMI8226 cell lines. Mechanistically, we show that loss of elongator complex-mediated downregulation of IRE1-XBP1 axis leads to stabilization of MCL1 and upregulation of BCL-XL and NOXA expression. We further show that upon treatment with MCL1i, KMS11-ELP4-KO cells have less disruption of MCL1:Bim complex and an increase in BCL-XL:Bim complex as compared with KMS11-NTC cells. The net increase in pro-survival MCL1 and BCL-XL proteins in ELP4-KO cells resulting in lower levels of unsequestered BIM upon AZD5991 treatment suggests a reduction in apoptotic priming.

The mechanistic link between the elongator complex and ER stress response pathway led us to test ER stress inducing drugs in these cell lines. We observed that ELP4-KO results in increased resistance to proteasome inhibitor Bortezomib and other ER stress inducers like Tunicamycin, Thapsigargin, and BrefeldinA as a monotherapy or in combination with AZD5991. These data are consistent with our hypothesis that ELP4-KO cells have reduced apoptotic priming and are thus multi-drug resistant.

As bortezomib is used in the clinic to treat MM patients, we asked if an elongator gene signature could be used to predict response to current therapies. We show that the elongator complex components could be used as a gene signature to stratify overall survival in MM patients (MMRF CoMMpass dataset). Moreover, ER stress response gene signature has been shown to be repressed in drug-resistant MM. Taken together, an integrated elongator and IRE-XBP1 gene signature could be a strong predictor of therapy response in MM .