Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia.

BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

DDB2 promotes melanoma cell growth by transcriptionally regulating the expression of KMT2A and predicts a poor prognosis.

Identification of potential key targets of melanoma, a fatal skin malignancy, is critical to the development of new cancer therapies. Lysine methyltransferase 2A (KMT2A) promotes melanoma growth by activating the human telomerase reverse transcriptase (hTERT) signaling pathway; however, the exact mechanism remains elusive. This study aimed to reveal new molecular targets that regulate KMT2A expression and melanoma growth. Using biotin-streptavidin-agarose pull-down and proteomics, we identified Damage-specific DNA-binding protein 2 (DDB2) as a KMT2A promoter-binding protein in melanoma cells and validated its role as a regulator of KMT2A/hTERT signaling. DDB2 knockdown inhibited the expression of KMT2A and hTERT and inhibited the growth of melanoma cells in vitro. Conversely, overexpression of DDB2 activated the expression of KMT2A and promoted the growth of melanoma cells. Additionally, we demonstrated that DDB2 expression was higher in tumor tissues of patients with melanoma than in corresponding normal tissues and was positively correlated with KMT2A expression. Kaplan-Meier analysis showed a poor prognosis in patients with high levels of DDB2 and KMT2A. Overall, our data suggest that DDB2 promotes melanoma cell growth through the transcriptional regulation of KMT2A expression and predicts poor prognosis. Therefore, targeting DDB2 may regulate the effects of KMT2A on melanoma growth and progression, providing a new potential therapeutic strategy for melanoma.

ASXLs binding to the PHD2/3 fingers of MLL4 provides a mechanism for the recruitment of BAP1 to active enhancers.

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.

Discovery of NSD2 non-histone substrates and design of a super-substrate.

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time.

MAIN CONCLUSION: In Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression. CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

SPA inhibits hBMSC osteogenic differentiation and M1 macrophage polarization by suppressing SETD2 in acute suppurative osteomyelitis.

To clarify the impact of SETD2 on macrophage function in pediatric patients with acute suppurative osteomyelitis and to elucidate the precise underlying mechanism. To gain insights into the potential functions of SETD2, a comprehensive study was conducted utilizing a co-culture model of human bone mesenchymal stem cells (hBMSCs) and bone marrow-derived macrophages (THP-1). A range of techniques were employed, including quantitative polymerase chain reaction, western blotting, ELISA, alkaline phosphatase activity assays, alizarin red S staining, luciferase reporter gene assays, and chromatin immunoprecipitation, to unravel the intricate interactions and molecular mechanisms involving SETD2 in this system. It was observed that SETD2 expression was reduced in THP-1 cells stimulated by staphylococcal protein A (SPA). Furthermore, the downregulation of SETD2 resulted in elevated M1 macrophage polarization and glycolysis, effects that were mitigated by SPA stimulation. Notably, SPA-stimulated THP-1 cells exhibited an increase in HIF-1α expression, which exhibited an inverse correlation with SETD2 levels. Moreover, it was discovered that SETD2 functioned as a catalyst for H3K36me3 and bound to the HIF-1α gene, which, in turn, regulated HIF-1α expression. Furthermore, the suppression of HIF-1α abrogated the consequences of SETD2 downregulation on glycolysis and M1 macrophage polarization. Lastly, the study demonstrated that M1 macrophage polarization serves as a mediator for BMP4's inhibitory effect on osteogenic differentiation of hBMSCs. This research has uncovered a previously unknown role of SETD2 in macrophages during osteomyelitis, revealing its significance in the pathogenesis of this condition. These findings suggest SETD2 as a novel target for the treatment of osteomyelitis.

PR-SET7 epigenetically restrains uterine interferon response and cell death governing proper postnatal stromal development.

The differentiation of the stroma is a hallmark event during postnatal uterine development. However, the spatiotemporal changes that occur during this process and the underlying regulatory mechanisms remain elusive. Here, we comprehensively delineated the dynamic development of the neonatal uterus at single-cell resolution and characterized two distinct stromal subpopulations, inner and outer stroma. Furthermore, single-cell RNA sequencing revealed that uterine ablation of Pr-set7, the sole methyltransferase catalyzing H4K20me1, led to a reduced proportion of the inner stroma due to massive cell death, thus impeding uterine development. By combining RNA sequencing and epigenetic profiling of H4K20me1, we demonstrated that PR-SET7-H4K20me1 either directly repressed the transcription of interferon stimulated genes or indirectly restricted the interferon response via silencing endogenous retroviruses. Declined H4K20me1 level caused viral mimicry responses and ZBP1-mediated apoptosis and necroptosis in stromal cells. Collectively, our study provides insight into the epigenetic machinery governing postnatal uterine stromal development mediated by PR-SET7.

Methylation of ESCRT-III components regulates the timing of cytokinetic abscission.

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.

KMT2D regulates activation, localization, and integrin expression by T-cells.

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8+ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4+ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.

The EV71 2A protease occupies the central cleft of SETD3 and disrupts SETD3-actin interaction.

SETD3 is an essential host factor for the replication of a variety of enteroviruses that specifically interacts with viral protease 2A. However, the interaction between SETD3 and the 2A protease has not been fully characterized. Here, we use X-ray crystallography and cryo-electron microscopy to determine the structures of SETD3 complexed with the 2A protease of EV71 to 3.5 Å and 3.1 Å resolution, respectively. We find that the 2A protease occupies the V-shaped central cleft of SETD3 through two discrete sites. The relative positions of the two proteins vary in the crystal and cryo-EM structures, showing dynamic binding. A biolayer interferometry assay shows that the EV71 2A protease outcompetes actin for SETD3 binding. We identify key 2A residues involved in SETD3 binding and demonstrate that 2A's ability to bind SETD3 correlates with EV71 production in cells. Coimmunoprecipitation experiments in EV71 infected and 2A expressing cells indicate that 2A interferes with the SETD3-actin complex, and the disruption of this complex reduces enterovirus replication. Together, these results reveal the molecular mechanism underlying the interplay between SETD3, actin, and viral 2A during virus replication.

Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6.

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.

Decreased serum levels of SETD1A protein in patients with schizophrenia.

OBJECTIVE: SET domain-containing protein 1A (SETD1A) histone lysine N-methyltransferase may serve as a biomarker for the auxiliary diagnosis and treatment assessment of schizophrenia (SCZ). The aim of this study was to compare serum levels of SETD1A protein between patients with SCZ and health controls. METHODS: Patients with SCZ and health controls were recruited from the Sixth Hospital of Changchun and the 'Survey on Chronic Diseases and Risk Factors among Adults in Jilin Province', respectively. The quantifications of lysine N-methyltransferase in peripheral serum were conducted by the ELISA method, and data was analyzed using the R software. RESULTS: Forty patients with SCZ (mean age: 33.97 ± 5.99 years) and forty healthy controls (mean age: 39.07 ± 4.62 years) were included. There was significantly lower concentration of SETD1A protein in the SCZ group compared with the control group (P < 0.001). This significant difference still exists after stratification by sex (P < 0.05). CONCLUSION: Our study demonstrates that decreased levels of serum SETD1A protein may be utilized as a possible peripheral biomarker for schizophrenia.

Suv39h-catalyzed H3K9me3 is critical for euchromatic genome organization and the maintenance of gene transcription.

H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.

Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts.

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

The NTE domain of PTENα/ß promotes cancer progression by interacting with WDR5 via its SSSRRSS motif.

PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.

[Phenotypic and genotypic spectrum of KMT2B dystonia. Description of three Colombian patients]. / Espectro fenotípico y genotípico de la distonía causada por el gen KMT2B. Descripción de tres casos colombianos.

INTRODUCTION: KMT2B-related dystonia is a childhood-onset movement disorder characterized by focal dystonia of the lower extremities progressing to generalized dystonia with predominant cervical, cranial, and laryngeal involvement. So far, fewer than 100 variants have been reported, the vast majority being de novo mutations. The presenting frame of KMT2B dystonia, with dysmorphology features and other complex neurologic symptoms shows the spectrum of KMT2B dystonia as a probable syndromic disease, rather than an isolated early-onset dystonia, which has been the classic view of the condition. CASE REPORTS: We report three patients who presented regression in their neurodevelopment, focal dystonia of the lower limbs with subsequent generalization, in whom two de novo variants were reported in the KMT2B gene, with a mean age of presentation lower than the average reported worldwide. CONCLUSIONS: We describe the largest local series of patients with KMT2B dystonia in Colombia (to our knowledge), which allows us to expand the genotype-phenotype relationship of this genetic dystonia. Although many affected individuals follow a similar disease course, it is important to determine clinical features that may be associated such as neurodevelopmental regression.

TITLE: Espectro fenotípico y genotípico de la distonía causada por el gen KMT2B. Descripción de tres casos colombianos.Introducción. La distonía relacionada con KMT2B es un trastorno del movimiento hipercinético, de inicio en la infancia, caracterizado inicialmente por distonía focal de las extremidades inferiores que progresa a una distonía generalizada con mayor afectación cervical, craneal y laríngea. Hasta el momento se han descrito aproximadamente 100 variantes causantes de enfermedad, y la mayoría son de novo. La presentación clínica de la distonía con hallazgos fenotípicos comunes en los pacientes, asociada a frecuente afectación del neurodesarrollo, afectación intelectual y otros trastornos del movimiento, hace pensar más en el espectro KMT2B como una condición sindrómica, más que en una distonía aislada de inicio temprano, como clásicamente se ha clasificado. Casos clínicos. Se presenta el caso de tres pacientes con regresión del neurodesarrollo, distonía focal de los miembros inferiores con posterior generalización, en quienes se identificaron tres variantes en el gen KMT2B, en dos de los casos de novo (en una de ellas se desconoce la segregación), y dos de ellas comunicadas por primera vez en la bibliografía. La edad media de presentación fue menor a la edad promedio notificada a nivel mundial. Conclusiones. Describimos una serie de pacientes colombianos con distonía DYT-KMT2B (la más grande en nuestro conocimiento a nivel local), lo que nos permite ampliar la relación genotipo-fenotipo de esta distonía genética. Si bien múltiples individuos afectados siguen un curso de enfermedad similar, es importante determinar las características clínicas que pueden estar asociadas, como la regresión del neurodesarrollo y su potencial clasificación como distonía compleja. Describimos, además, dos nuevas variantes asociadas al fenotipo.

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitor (PARPi) resistance poses a significant challenge in ovarian carcinoma (OC). While the role of DOT1L in cancer and chemoresistance is acknowledged, its specific role in PARPi resistance remains unclear. This study aims to elucidate the molecular mechanism of DOT1L in PARPi resistance in OC patients. METHODS: This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance. RESULTS: Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs). CONCLUSIONS: Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients.

Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.