Expanded profiling of WD repeat domain 5 inhibitors reveals actionable strategies for the treatment of hematologic malignancies.

WD40 Repeat Domain 5 (WDR5) is a highly conserved nuclear protein that recruits MYC oncoprotein transcription factors to chromatin to stimulate ribosomal protein gene expression. WDR5 is tethered to chromatin via an arginine-binding cavity known as the "WIN" site. Multiple pharmacological inhibitors of the WDR5-interaction site of WDR5 (WINi) have been described, including those with picomolar affinity and oral bioavailability in mice. Thus far, however, WINi have only been shown to be effective against a number of rare cancer types retaining wild-type p53. To explore the full potential of WINi for cancer therapy, we systematically profiled WINi across a panel of cancer cells, alone and in combination with other agents. We report that WINi are unexpectedly active against cells derived from both solid and blood-borne cancers, including those with mutant p53. Among hematologic malignancies, we find that WINi are effective as a single agent against leukemia and diffuse large B cell lymphoma xenograft models, and can be combined with the approved drug venetoclax to suppress disseminated acute myeloid leukemia in vivo. These studies reveal actionable strategies for the application of WINi to treat blood-borne cancers and forecast expanded utility of WINi against other cancer types.

Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC.

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.

HER2-Directed Therapy in Advanced Gastric and Gastroesophageal Adenocarcinoma: Triumphs and Troubles.

OPINION STATEMENT: Gastric and gastroesophageal junction (GEJ) cancers represent the third leading cause of malignancy-associated death worldwide. Approximately 15-20% of these adenocarcinomas overexpress the human epidermal growth factor receptor 2 (HER2), a pro-proliferative receptor tyrosine kinase that has been therapeutically exploited in other disease contexts. The landmark ToGA trial demonstrated that trastuzumab, an anti-HER2 antibody, could improve overall survival for patients with HER2 overexpressing advanced gastric and GEJ adenocarcinomas. In the ensuing decade, great effort has been made to refine and expand this therapeutic strategy through a variety of avenues including optimization of chemotherapy backbones, identifying potential synergy with immune checkpoint inhibition, deployment of alternative HER2-targeted antibodies, use of small molecule inhibitors, and development of HER2-directed antibody drug conjugates. While the results of these efforts have had variable success, they have led to a greater understanding of the mechanisms of both primary and acquired resistance to HER2-directed therapies, laying the groundwork for future investigations. Recently, KEYNOTE-811 and DESTINY-Gastric01 have led to the FDA approvals of pembrolizumab in combination with trastuzumab and chemotherapy in the 1st-line advanced setting and trastuzumab deruxtecan (fam-trastuzumab deruxtecan-nxki) in the 2nd-line setting, respectively. Herein, we review these significant works as well as discuss the ongoing investigations they have inspired, which aim to find and utilize additional means for targeting HER2 in gastric and GEJ cancers.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the "WIN" site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small molecule WIN site inhibitors, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anti-cancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anti-cancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

New Opportunities for Minimizing Toxicity in Rectal Cancer Management.

Advances in multimodal management of locally advanced rectal cancer (LARC), consisting of preoperative chemotherapy and/or radiotherapy followed by surgery with or without adjuvant chemotherapy, have improved local disease control and patient survival but are associated with significant risk for acute and long-term morbidity. Recently published trials, evaluating treatment dose intensification via the addition of preoperative induction or consolidation chemotherapy (total neoadjuvant therapy [TNT]), have demonstrated improved tumor response rates while maintaining acceptable toxicity. In addition, TNT has led to an increased number of patients achieving a clinical complete response and thus eligible to pursue a nonoperative, organ-preserving, watch and wait approach, thereby avoiding toxicities associated with surgery, such as bowel dysfunction and stoma-related complications. Ongoing trials using immune checkpoint inhibitors in patients with mismatch repair-deficient tumors suggest that this subgroup of patients with LARC could potentially be treated with immunotherapy alone, sparing them the toxicity associated with preoperative treatment and surgery. However, the majority of rectal cancers are mismatch repair-proficient and less responsive to immune checkpoint inhibitors and require multimodal management. The synergy noted in preclinical studies between immunotherapy and radiotherapy on immunogenic tumor cell death has led to the design of ongoing clinical trials that explore the benefit of combining radiotherapy, chemotherapy, and immunotherapy (mainly of immune checkpoint inhibitors) and aim to increase the number of patients eligible for organ preservation.

MTBP and MYC: A Dynamic Duo in Proliferation, Cancer, and Aging.

The oncogenic transcription factor c-MYC (MYC) is highly conserved across species and is frequently overexpressed or dysregulated in human cancers. MYC regulates a wide range of critical cellular and oncogenic activities including proliferation, metabolism, metastasis, apoptosis, and differentiation by transcriptionally activating or repressing the expression of a large number of genes. This activity of MYC is not carried out in isolation, instead relying on its association with a myriad of protein cofactors. We determined that MDM Two Binding Protein (MTBP) indirectly binds MYC and is a novel MYC transcriptional cofactor. MTBP promotes MYC-mediated transcriptional activity, proliferation, and cellular transformation by binding in a protein complex with MYC at MYC-bound promoters. This discovery provided critical context for data linking MTBP to aging as well as a rapidly expanding body of evidence demonstrating MTBP is overexpressed in many human malignancies, is often linked to poor patient outcomes, and is necessary for cancer cell survival. As such, MTBP represents a novel and potentially broad reaching oncologic drug target, particularly when MYC is dysregulated. Here we have reviewed the discovery of MTBP and the initial controversy with its function as well as its associations with proliferation, MYC, DNA replication, aging, and human cancer.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

Evolving Landscape of Systemic Therapy for Hepatocellular Carcinoma: Breakthroughs, Toxicities, and Future Frontiers.

The incidence and death rates of hepatocellular carcinoma (HCC) are rising. For more than a decade, the multikinase inhibitor sorafenib was the only U.S. Food and Drug Administration (FDA)-approved systemic therapy for HCC. However, since 2017, five additional agents have been approved in the first- or second-line setting. Although this represents an incredible victory for the field, there are no clear guidelines for agent selection on the basis of either patient or tumor characteristics. Here, we review the available systemic therapy options for advanced HCC and reported clinical data for each. We outline each agent's unique toxicity profile, potential impact on patient quality of life, monitoring recommendations, and supportive strategies. Last, we review molecular and immunologic classifications of HCC as well as preclinical data that may serve as a basis for future biomarker enriched clinical trials to enable precision oncology care in HCC.

Haploinsufficiency of the Myc regulator Mtbp extends survival and delays tumor development in aging mice.

Alterations of specific genes can modulate aging. Myc, a transcription factor that regulates the expression of many genes involved in critical cellular functions was shown to have a role in controlling longevity. Decreased expression of Myc inhibited many of the deleterious effects of aging and increased lifespan in mice. Without altering Myc expression, reduced levels of Mtbp, a recently identified regulator of Myc, limit Myc transcriptional activity and proliferation, while increased levels promote Myc-mediated effects. To determine the contribution of Mtbp to the effects of Myc on aging, we studied a large cohort of Mtbp heterozygous mice and littermate matched wild-type controls. Mtbp haploinsufficiency significantly increased longevity and maximal survival in mice. Reduced levels of Mtbp did not alter locomotor activity, litter size, or body size, but Mtbp heterozygous mice did exhibit elevated markers of metabolism, particularly in the liver. Mtbp+/- mice also had a significant delay in spontaneous cancer development, which was most prominent in the hematopoietic system, and an altered tumor spectrum compared to Mtbp+/+ mice. Therefore, the data suggest Mtbp is a regulator of longevity in mice that mimics some, but not all, of the properties of Myc in aging.

MTBP is overexpressed in triple-negative breast cancer and contributes to its growth and survival.

UNLABELLED: Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with estrogen receptor-positive (ER(+)) and HER2(+) breast cancer. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. MTBP (MDM2-binding protein), a gene linked to cellular proliferation and a transcriptional target of the MYC oncogene, is overexpressed in human malignancies, yet its contribution to cancer remains unresolved. Evaluation of mRNA expression and copy number variation data from The Cancer Genome Atlas (TCGA) revealed that MTBP is commonly overexpressed in breast cancer and 19% show amplification of MTBP. Increased transcript or gene amplification of MTBP significantly correlated with reduced breast cancer patient survival. Further analysis revealed that while MTBP mRNA is overexpressed in both ER(+) and HER2(+) breast cancers, its expression is highest in TNBC. MTBP mRNA and protein levels were also significantly elevated in a panel of human TNBC cell lines. Knockdown of MTBP in TNBC cells induced apoptosis and significantly reduced TNBC cell growth and soft agar colony formation, which was rescued by expression of shRNA-resistant Mtbp. Notably, inducible knockdown of MTBP expression significantly impaired TNBC tumor growth, in vivo, including in established tumors. Thus, these data emphasize that MTBP is important for the growth and survival of TNBC and warrants further investigation as a potential novel therapeutic target. IMPLICATIONS: MTBP significantly contributes to breast cancer survival and is a potential novel therapeutic target in TNBC.

Oncogenic protein MTBP interacts with MYC to promote tumorigenesis.

Despite its involvement in most human cancers, MYC continues to pose a challenge as a readily tractable therapeutic target. Here we identify the MYC transcriptional cofactors TIP48 and TIP49 and MYC as novel binding partners of Mdm2-binding protein (MTBP), a functionally undefined protein that we show is oncogenic and overexpressed in many human cancers. MTBP associated with MYC at promoters and increased MYC-mediated transcription, proliferation, neoplastic transformation, and tumor development. In breast cancer specimens, we determined overexpression of both MYC and MTBP was associated with a reduction in 10-year patient survival compared with MYC overexpression alone. MTBP was also frequently co-amplified with MYC in many human cancers. Mechanistic investigations implicated associations with TIP48/TIP49 as well as MYC in MTBP function in cellular transformation and the growth of human breast cancer cells. Taken together, our findings show MTBP functions with MYC to promote malignancy, identifying this protein as a novel general therapeutic target in human cancer.