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Ribosome Depletion and p53 Axis Activation Drive the Response of MLL-Rearranged Cancer Cells to WDR5 Inhibition

Core Concepts
WIN site inhibitors of the chromatin-associated protein WDR5 induce depletion of the ribosome inventory, leading to a broad translational choke and activation of the p53-MDM4 axis, which drives apoptosis in MLL-rearranged cancer cells.
The content describes an integrated multi-omics approach to investigate the mechanism of action of WDR5 WIN site inhibitors (WINi) in MLL-rearranged (MLLr) cancer cells. Key highlights: WINi with improved potency (C16) have similar transcriptional effects as earlier compounds (C6), suppressing expression of ribosomal protein genes (RPGs) bound by WDR5. At the translational level, WINi broadly reduce the efficiency of mRNA translation, beyond just the WDR5-bound RPGs. This translates to a progressive depletion of the entire ribosome inventory, accompanied by nucleolar stress and activation of the p53 pathway. The p53 response is driven in part by RPL22-dependent alternative splicing of the p53 antagonist MDM4. A CRISPR screen identifies p53, BCL-2/BAX, and BRD3 as modulators of the response to WINi. WINi synergize with agents like venetoclax, mivebresib, and PRMT5 inhibitors, suggesting optimal clinical implementation will involve combination therapies. Overall, the study reveals a novel mechanism of action for WINi in MLLr cells, centered on ribosome depletion and p53 axis activation, and provides a resource to guide their clinical development.
"WDR5 is a highly-conserved protein that moonlights in a variety of functions in the nucleus." "WIN site inhibitors (WINi) have been described, including those that are orally bioavailable and have anti-tumor activity in vivo." "We observe that maximal suppression of RPG transcripts occurs at ∼2 μM for C6 and ∼100 nM for C16 in MV4;11 and MOLM13 cells." "Between ∼4,500 (C16) and ∼5,900 (C6) transcripts show decreased translation efficiency in response to WINi." "Almost all ribosomal subunits are in deficit after 72 hours of C16 treatment, regardless of whether or not they are encoded by a WDR5-bound gene." "The most highly-enriched gene in the CRISPR screen is TP53."
"WIN site inhibitors kill MLLr cells via depletion of part of the ribosome inventory that induces apoptosis via a ribosome biogenesis stress response." "Contrary to our earlier idea that WINi promote ribosome subunit imbalance, however, these data support a simpler model in which these inhibitors ultimately induce attrition of the majority of ribosomal proteins—as well as mature rRNAs." "Collectively, these findings demonstrate functional involvement of the ribosomal protein RPL22 in the response to WINi, and confirm the importance of the p53 network to robust inhibition of MLLr cell growth by these agents."

Deeper Inquiries

How might the selective depletion of ribosomal proteins impact other cellular processes beyond translation and p53 activation?

The selective depletion of ribosomal proteins induced by WINi can have far-reaching effects on various cellular processes beyond translation and p53 activation. Firstly, ribosomal proteins play crucial roles in ribosome biogenesis, and their depletion can disrupt the assembly of functional ribosomes, leading to a decrease in protein synthesis efficiency. This can result in a global slowdown of cellular processes that rely on the timely production of proteins, impacting cell growth, proliferation, and overall metabolism. Secondly, ribosomal proteins have been implicated in regulating cell cycle progression and cell division. Depletion of ribosomal proteins can disrupt the balance between cell growth and division, potentially leading to cell cycle arrest or aberrant cell division. This dysregulation can have implications for cell viability and contribute to the anti-proliferative effects of WINi. Furthermore, ribosomal proteins have been linked to cellular stress responses, including the unfolded protein response (UPR) and autophagy. Depletion of ribosomal proteins can activate stress signaling pathways, such as the UPR, to cope with the imbalance in protein synthesis machinery. This can trigger downstream effects on cellular homeostasis and survival mechanisms. Additionally, ribosomal proteins have been implicated in DNA repair processes and maintenance of genomic stability. Their depletion can compromise DNA repair mechanisms, leading to an accumulation of DNA damage and genomic instability. This can have implications for the overall genomic integrity of the cell and potentially contribute to the cytotoxic effects of WINi. In summary, the selective depletion of ribosomal proteins induced by WINi can impact a wide range of cellular processes beyond translation and p53 activation, including cell growth, proliferation, stress responses, cell cycle regulation, DNA repair, and genomic stability.

What are the potential mechanisms by which resistance to WINi could emerge, beyond the pathways identified in the CRISPR screen?

Resistance to WINi can potentially emerge through various mechanisms beyond those identified in the CRISPR screen. Some potential mechanisms of resistance include: Activation of alternative survival pathways: Cancer cells may activate alternative survival pathways to bypass the effects of WINi-induced ribosome depletion. This could involve upregulation of compensatory pathways that promote cell survival and proliferation independent of the ribosome biogenesis stress response. Genetic mutations: Cancer cells may acquire genetic mutations that confer resistance to WINi. These mutations could affect the target binding site of WINi on WDR5 or alter downstream signaling pathways involved in the response to ribosome depletion. Epigenetic changes: Epigenetic modifications, such as alterations in chromatin structure or histone modifications, could impact the sensitivity of cancer cells to WINi. Changes in the epigenetic landscape may modulate the expression of genes involved in the response to ribosome depletion, leading to resistance. Activation of drug efflux pumps: Cancer cells may upregulate drug efflux pumps to actively pump out WINi from the cells, reducing their intracellular concentration and efficacy. This mechanism can contribute to decreased drug accumulation and resistance to treatment. Metabolic reprogramming: Cancer cells may undergo metabolic reprogramming to adapt to the stress induced by WINi treatment. Alterations in cellular metabolism can provide energy and resources for cell survival and proliferation, counteracting the effects of ribosome depletion. Tumor microenvironment interactions: Interactions with the tumor microenvironment, such as communication with stromal cells or immune cells, can influence the response of cancer cells to WINi. Changes in the tumor microenvironment may create a protective niche that promotes resistance to treatment. Overall, resistance to WINi can arise through a combination of genetic, epigenetic, metabolic, and microenvironmental factors that enable cancer cells to evade the effects of ribosome depletion and continue to proliferate.

Given the broad effects of WINi on the ribosome inventory, what other cancer types or contexts beyond MLL-rearranged leukemia might these inhibitors be effective against?

The broad effects of WINi on the ribosome inventory suggest that these inhibitors may be effective against various cancer types beyond MLL-rearranged leukemia. Some potential cancer types or contexts where WINi could be effective include: MYC-driven cancers: WINi have shown efficacy in MYC-driven cancers, where MYC overexpression leads to increased ribosome biogenesis and protein synthesis. Targeting the interaction between WDR5 and MYC could disrupt this process and inhibit the growth of MYC-driven tumors. Neuroblastomas: WINi have demonstrated activity in neuroblastomas, a type of childhood cancer characterized by MYCN amplification. By targeting the WDR5-MYC interaction, WINi could disrupt MYCN-driven oncogenic pathways and inhibit neuroblastoma growth. Metastatic breast cancers: WINi have shown promise in metastatic breast cancers, where WDR5 overexpression is associated with aggressive disease. Inhibiting WDR5 could suppress the growth and metastasis of breast cancer cells. C/EBPα-mutant leukemias: C/EBPα-mutant leukemias, characterized by mutations in the C/EBPα gene, may also be sensitive to WINi. WDR5 inhibition could disrupt the aberrant transcriptional programs driven by mutant C/EBPα and inhibit leukemia cell growth. Rhabdoid tumors: WINi have demonstrated activity in rhabdoid tumors, a rare and aggressive type of pediatric cancer. By targeting WDR5, WINi could disrupt the oncogenic pathways driving rhabdoid tumor growth and provide a therapeutic option for these patients. Overall, the broad mechanism of action of WINi on ribosome biogenesis and protein synthesis suggests that these inhibitors may have efficacy in a variety of cancer types characterized by dysregulated ribosome function and protein synthesis. Further preclinical and clinical studies are needed to explore the potential of WINi in these diverse cancer contexts.