Novel NOTCH1 Effector NFIL3 Is a Therapeutic Target to Combat Drug Resistance and Improve Patient Outcomes in Newly Diagnosed and Relapsed-Refractory Multiple Myeloma

Aberrant activation of Notch signaling contributes to drug resistance and induces bone resorption and destruction in multiple myeloma (MM), posing a significant challenge in achieving lasting remissions and increasing the risk of relapse. Biochemical inhibition of the Notch pathway through γ-secretase inhibitors leads to secretory goblet cell metaplasia and severe gastrointestinal side effects, when used in clinical trials. Thus, there is a need for novel druggable Notch effectors that can combat drug resistance in MM while minimizing toxicity.

To this end, transcriptome profiling was conducted on human MM.1S cells following NOTCH1 knockdown or inhibition, identifying 40 gene candidates based on the strength and consistency of regulation (log2FC; padj<0.05). Subsequently, a focused short hairpin RNA (shRNA) library was established and screened in NOTCH1-overexpressing MM cells (U266-N1IC, U266-N1ΔE) under the selective pressure of lenalidomide (Len), melphalan (Mel), or bortezomib (Btz). Genes were identified as high-potential targets if their corresponding shRNAs were significantly depleted in the remaining drug-resistant cell clones. Genes known to be involved in the NOTCH or Wnt pathways, which are relevant to disease progression, such as HES1, HEY1, and WNT5B were found among the top hits from the screen. Notably, the results demonstrated that the upregulation of the nuclear factor interleukin 3 regulated (NFIL3) conferred resistance to the drugs used in MM cells. ChIP-seq analysis illustrates the recruitment of RBPJ to NFIL3 locus among the upstream region and around the transcription start site, confirming NFIL3 is a direct NOTCH1 target.

Hindering NFIL3 expression through shRNA knockdown or using the oleanane-type triterpenoid saponin Raddeanin A (RA) presented decreased expression of NOTCH-related genes and inhibition of the JAK-STAT3-NFIL3 signaling pathway. Immunoblotting revealed decreased BCL-2 protein expression and increased cleaved Caspase-3 levels following NFIL3 inhibition, indicating altered BCL-2-dependent apoptosis resistance. Slowed cell growth and reduced cell viability could be observed under RA treatment combined with additional drugs in vitro with MM cell lines and in vivo with murine MM model, showing RA enhances the effect of Len, Mel, or Btz for controlling tumor growth. Additionally, RA also displays the capability to prevent osteolytic bone lesion by blocking osteoclast development and activity in vivo. In patients with newly diagnosed and relapsed-refractory MM, RA was effective against primary MM cells regardless of previous therapy and increased their sensitivity to other drugs, suggesting its potential to counteract drug resistance in the early and late stages of the disease. In addition, we developed a biopolymer-based nanoformulation of RA with high drug loading capacity for in vivo applications, which significantly improves compound solubility and minimizes toxicity in vivo.

In conclusion, we identified upregulation of NFIL3 as a novel mechanism for drug resistance in relapsed-refractory MM. The high expression of NFIL3 drives a vicious cycle to maintain aberrant NOTCH1 activation. Therefore, targeting NFIL3 in MM eliminates high NOTCH1 activity and could control drug resistance. Ultimately, our findings may lead to new strategies to overcome the drug resistance in MM by utilizing inhibitors of the JAK-STAT3-NFIL3 pathway, hence avoiding the off-target intestinal effects of direct NOTCH inhibition and improving MM patient outcomes.