Mixed Lineage Leukemia Research Articles

Septin11 and Septin 8 Play Critical and Overlapping Roles Murine Stem Cell Function

Septins are highly conserved GTP binding proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis and are now considered the fourth component of the cytoskeleton. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previous data in our laboratory led to the discovery of a small molecular inhibitor, DW0254, which inhibits RAS function and its downstream target RhoGTPase, Rac, including the hematopoietic-specific Rac2 GTPase, also implicated in MLL leukemias. The inhibitor leads to apoptosis of leukemia cell lines. Using biophysical methods, we discovered that DW0254 binds both the RAS chaperone, PDE6D, and Septin11, which is a member of the Septin6 family (Sara CN, et al., Blood Cancer 2022). Although Septins have been reported to be involved in function of the RhoGTPase Cdc42 and hematopoietic stem progenitor cell (HSPC) polarity, the role of Septin11 in hematopoiesis has not been reported. Septin11 is a member of the Septin6 family of proteins which consists of Septin6, 8, 10, 11 and 14 with significant homology between family members. Previous data in yeast has hypothesized functional overlap within each family of Septin proteins. To better understand the functional role of Septin11, we created lentivirus vectors tagged with a GFP reporter that contained shmiRs against Septin11. Culture of Septin11 knockdown (Septin11 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 13-fold decrease in cell counts compared to controls (p=0.02) and a decrease in total clonogenic activity (Figure 1) with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies. Reduced clonogenic numbers were accompanied by a significant increase in early apoptotic cells suggesting that Septin11 is important in regulating apoptosis in HSPC. To determine the potential role of Septin11 in more primitive hematopoietic cells, we utilized competitive repopulation assays. Transduced BoyJ HSPC (GFP+ Septin11 KD and BFP+ control, mixed 1:1) were injected into lethally irradiated C57Bl/6 mice. We observed a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary transplant recipients after knockdown of Septin11 (Table 1). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin11 most clearly in LT-HSC and ST-HSC (Table 1) with no statistically significant change in MPP. Therefore, to better study the hematopoietic stem cell compartment, we performed secondary transplants utilizing the cells obtained from primary competitive repopulation assays. We observed an unexpected recovery in engraftment in secondary transplant recipients that was accompanied by an increase in the mRNA expression of Septin8. Since Septin8 and Septin11 are closely related we hypothesized that induction of Septin8 was subserving key functions of Septin11. To test this hypothesis, we created a lentivirus vector that expressed both a shmiR against Septin11 and the Septin8 cDNA. Concurrent overexpression of Septin8 with Septin11 knockdown HSPC caused a significant increase in the total number of colonies compared to controls (Figure 1). These data indicate an important role of Septin11 in murine hematopoiesis and a potential redundant role for Septin8.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

Prognostic Value of Copy Number Aberrations and Copy Neutral Loss of Heterozygosity in Acute Myeloid Leukemia

Background: Chromosomal abnormalities are key to the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Chromosomal genomic array testing (CGAT) can identify copy neutral loss of heterozygosity (cnLOH) and genomic copy number alterations (CNAs) that are undetected by conventional karyotype or fluorescence in situ hybridization (FISH). Previous studies have shown that cnLOH is correlated with a shorter duration of complete remission (CR) and worse overall survival (OS) in AML (Grosneth et. al, Cancer, 2015). The primary objective of this exploratory study is to examine the predictive power of CGAT in a cohort of AML patients with majority normal karyotype. Methods: CGAT analyses were collected from 187 subjects with AML or other high grade myeloid neoplasm (myeloid blasts >10% in peripheral blood or bone marrow) between 2012 and 2022 at Fred Hutchinson Cancer Center. CGAT utilized CytoScan™ HD Array (ThermoFisher, MA), targeting genome-wide region with 2.4 million markers for copy number and approximately 750,000 genotype-able SNPs. The data were analyzed using the Nexus Copy Number software with manual curation. Based on the assay validation in our CLIA-certified diagnostic lab, the cutoff is 100 Kb for copy number aberrations and 10 Mb for cnLOH. Survival predictive power analysis (SPP), a log-rank based test using Nexus, was applied to identify aberrations significant for event free survival (EFS) and overall survival (OS). SPP analysis tabulated genomic segments based on p-values with the alpha value cutoff at 0.05. Using frequency as the filtering criteria (present in >5 patients), 132 SPP for EFS, and 73 SPP for OS were identified. SPP were then manually curated to (1) remove those representing constitutional absence of heterozygosity (AOH) and those overlapping with benign copy number variance and (2) connect adjacent SPP into contiguous regions. The presence and absence of the identified SPP were then tabulated, cleaned up, and used to generate Kaplan-Meier curves for EFS and OS. This process identified 5 SPP that are significant for EFS and 2 SPP for OS. A multivariate analysis was performed to determine the hazard ratio (HR) of these 5 genetic aberrations for EFS and OS, adjusted for patient age, secondary/treatment related AML, ELN 2022 risk, and receipt of allogeneic hematopoietic cell transplantation (HCT) as a time-varying covariate. Results: In our 187 subject cohort, the median age was 63 (range 20-87). 76 (41%) had secondary or treatment related disease. 97 (52%) went on to receive a hematopoietic stem cell transplant. 3 had favorable risk disease, 14 had adverse risk, and 170 had intermediate risk by karyotype and FISH using ELN 2022 criteria. 120 (64%) had normal karyotype. 5 subjects had 4q cnLOH, 6 had 5q ( EGR1) loss, 7 had 9p cnLOH, 10 had mixed-lineage leukemia gene partial tandem duplication ( MLL-PTD), and 4 had 7q deletion. Univariate analysis identified five SPP that are significant for EFS: 4q cnLOH (p=0.002), 5q ( EGR1) loss (p<0.001), 9p cnLOH (p<0.001), MLL-PTD (p<0.001), and 7q deletion (p<0.001). 2 SPP were significant for OS: 9p cnLOH (p=0.001) and MLL-PTD (p=0.036). Multivariate analysis for EFS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 3.5 for 4q cnLOH (p=0.008), 2.3 for 5q ( EGR1) loss (p=0.16), 4.5 for 7q deletion (p=0.007), 5.7 for 9p cnLOH (p<0.001), and 2.9 for MLL-PTD (p=0.002). Multivariate analysis for OS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 2.0 for 4q cnLOH (p=0.23), 0.5 for 5q ( EGR1) loss (p=0.36), 1.3 for 7q deletion (p=0.66), 4.9 for 9p cnLOH (p=0.001), and 2.3 for MLL-PTD (p=0.04). Discussion: This study has found 5 key genetic changes identifiable via CGAT that are significant for shorter EFS and/or OS in AML patients. Our next step is to validate these findings in a large independent cohort of AML patients. CGAT is a powerful adjunct to conventional karyotype and FISH for the detection of cnLOH and can be used to identify CNAs below the level of detection of karyotype. For these reasons, CGAT may be most useful in intermediate risk patients. Further research is warranted to fully understand the prognostic power of CGAT in AML.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

Epigenetically-driven persistence of inflammatory and oxidative phenotypes in adolescents born to women with gestational diabetes

Abstract Background According to the theory of developmental origins of health and disease1, environmental changes during the in-utero period can permanently affect health and vulnerability to diseases later in life. As a matter of fact, the offspring of women with gestational diabetes (GD) exhibit a higher risk for developing obesity, type 2 diabetes (T2D), and atherosclerotic cardiovascular disease (ASCVD) than those individuals with a family history of diabetes2–4. Importantly, epigenetics has emerged as a crucial determinant for this transmission across generations5–8. Mixed Lineage Leukemia 1 (MLL1) histone methyltransferase activates inflammatory and oxidative pathways through trimethylation of histone 3 at lysine 4 amino (H3K4me3)9. Purpose To investigate whether such epigenetic mark regulates genes triggering inflammation and oxidative stress in GD women, and its potential transmission and persistence in their offspring. Methods We isolated peripheral blood mononuclear cells (PBMCs), fetal cord blood mononuclear cells (CBMCs), and umbilical cord vein endothelial cells (HUVECs) from GD and control pregnant women, as well as PBMCs and plasma samples from a retrospective cohort of adolescents (12-16 years old) born to control or GD women. Gene and protein expressions were investigated by RT-PCR, immunoblotting and/or confocal microscopy. H3K4me3 levels on NF-kB p65 and NADPH oxidase isoform 4 (NOX4) promoter regions were assessed by chromatin immunoprecipitation (ChIP) and RT-PCR. Mechanistic studies were performed in control- and GD-HUVECs using the MLL1 inhibitor MM-102 to assess downstream inflammatory mediators and superoxide anion (O2-) generation by electron spin resonance (ESR) spectroscopy. Results Our data showed upregulation of MLL1 and H3K4me3 levels in PBMCs, HUVECs, and CBMCs isolated from GD as compared to control women. ChIP analysis revealed H3K4me3 enrichment in the promoter region of the inflammatory and oxidative master regulatory genes NF-kB p65 and NOX4, respectively, in these cells. We also found an upregulation of NF-kB p65 target genes IL-6, MCP-1, ICAM-1, and VCAM-1, and increased monocyte adhesion and O2- production. The inhibition of MLL1 was able to reduce H3K4me3 levels blunting the inflammatory and oxidative phenotypes in GD-HUVECs. Interestingly enough, we demonstrated a persistent increase of H3K4me3 in the NF-kB p65 promoter region of PBMCs together with an elevation of inflammatory markers in plasma from adolescents born to GD women. Conclusions Our results suggest that GD may induce inflammation and oxidative stress through MLL1-dependent H3K4me3 mechanisms. Such epigenetic mark is transmitted to the offspring and, most importantly, can still be found in adolescence as a pro-inflammatory trigger. These findings pave the way for pharmacological reprogramming of adverse histone modifications to counteract early abnormal phenotypes in GD offspring which may accelerate ASCVD burden.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 μM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 μM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

Komet-008: A Phase 1 Study to Determine the Safety and Tolerability of Ziftomenib Combinations for the Treatment of KMT2A-Rearranged or NPM1-Mutant Relapsed/Refractory Acute Myeloid Leukemia

Background and Significance: Relapsed or refractory (R/R) acute myeloid leukemia (AML) with nucleophosmin 1-mutations ( NPM1-m) or KMT2A-rearrangment ( KMT2A-r) remain a high unmet need. Ziftomenib is a potent and selective inhibitor of the interaction between menin and mixed-lineage leukemia (MLL) lysine[K]-specific methyltransferase 2A ( KMT2A), which drives leukemogenesis in these subtypes. Preclinical data have demonstrated ziftomenib's ability to target multiple types of menin-dependent AML clones, and that the antitumor activity of ziftomenib and other menin inhibitors in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models of KMT2A-r and NPM1-m AML is enhanced by the addition of FLT3 inhibitors such as gilteritinib and quizartinib. An ongoing Phase 1/2 study (KOMET-001) in R/R AML has demonstrated that ziftomenib monotherapy is safe and well tolerated and that the clinical activity at the recommended phase 2 dose (RP2D) is robust, as the rates of response were not affected by the presence of downstream co-mutations, such as FLT3 and IDH1 or IDH2. Given the monotherapy activity seen to date, the development of ziftomenib in combination with standard-of-care (SOC) chemotherapies or FLT3 inhibitors may provide increased clinical benefit for R/R AML patients with KMT2A-r or with NPM1-m with or without concurrent FLT3-mutations ( FLT3-m) and warrants further investigation. Study Design and Methods: KOMET-008 (NCT# 05735184) is an open-label, dose escalation and expansion study to determine the safety, tolerability, and preliminary efficacy of ziftomenib when combined with SOC regimens for the treatment of either NPM1-m (Arm A) or KMT2A-r (Arm B) R/R AML. Each genetically defined arm will evaluate ziftomenib in combination with various SOC treatments in separate and independent cohorts, as outlined below: Arm A:NPM1-m R/R AML Cohort A-1: Ziftomenib plus FLAG-IDACohort A-2: Ziftomenib plus LDACCohort A-3: Ziftomenib plus gilteritinib (for NPM1-m + FLT3-m R/R AML) Arm B: KMT2A-r R/R AML Cohort B-1: Ziftomenib plus FLAG-IDACohort B-2: Ziftomenib plus LDAC The Part 1a dose escalation uses a i3+3 design, followed by dose expansion/validation in Part 1b. Key eligibility criteria include adults (≥18 years) with documented NPM1-m (±co-occurring FLT3-m) or KMT2A-r R/R AML, ECOG ≤2, prior hematopoietic stem cell transplant (HSCT) allowed, if recovered and prior gilteritinib allowed. Patients who receive an HSCT during study participation will be allowed to receive ziftomenib maintenance therapy upon recovery. The primary objectives are to establish the safety and tolerability of ziftomenib when combined with SOC therapies (ie, dose limiting toxicities, AEs per CTCAE v5.0) and to identify the RP2D of ziftomenib for each SOC combination in each genetically defined cohort. Secondary endpoints include preliminary efficacy (including MRD-negative responses and number of patients able to receive stem cell transplant) and pharmacokinetics. Exploratory end points include biomarkers for efficacy and resistance and pharmacodynamic biomarkers potentially related to the activity of ziftomenib when combined with SOC treatments in adults with R/R AML with NPM1-m or KMT2A-r.

Early Results of the Phase I/II Study Investigating the All-Oral Combination of the Menin Inhibitor Revumenib (SNDX-5613) with Decitabine/Cedazuridine (ASTX727) and Venetoclax in Acute Myeloid Leukemia (SAVE)

Background: The interaction of menin with lysine methyltransferase 2A (KMT2A) is a dependency in acute leukemia caused by either rearrangement of the KMT2A (KMT2Ar) or Nucleoporin 98 ( NUP98r) genes, or mutation of the Nucleophosmin 1 gene ( NPM1mt). The menin inhibitor revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of the menin-KMT2A interaction with demonstrated safety and clinical activity in highly refractory acute leukemias (Issa GC, Nature 2023). KMT2Ar or NPM1mt leukemias are highly susceptible to induction of apoptosis through BCL2 inhibition, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021). Therefore, we designed a phase I/II, investigator-initiated trial of the all-oral combination of revumenib, venetoclax and the hypomethylating agent ASTX727 in children and adults with relapsed/refractory (R/R) acute myeloid leukemia (AML) (NCT05360160). Methods: Patients (pts) with R/R AML or myeloid mixed-lineage acute leukemia (MPAL) aged 12 years and older were eligible.Dose escalation followed a 3+3 design. ASTX727 (decitabine/ cedazuridine) was administered at 35 mg/100 mg PO daily days 1-5, venetoclax at 400 mg (target dose) PO daily days 1-14, and revumenib 113 mg PO Q12h (dose level [DL] 0) or 163 mg PO Q12h (DL 1, used in phase II monotherapy), days 1-28 with either posaconazole or voriconazole (strong CYP3A4 inhibitors, for antifungal prophylaxis). Maintenance with revumenib monotherapy is planned following hematopoietic stem cell transplant (HSCT) for 1 year. For the first pt cohort, bone marrow examination was performed on cycle 1 day 14 and at the end of the cycle to assess for early morphologic remission and to inform future dose adjustments. Results: As of 7/20/2023, 8 pts were enrolled, 6 at DL0, and 2 at DL1. The median age was 27 years (range, 12-62 years), including 2 children (ages 12 and 16), 5 with KMT2Ar, 2 with NUP98r and 1 with NPM1mt. One pt had MPAL, and one had bone marrow and extramedullary disease. The median prior lines of therapy was 2.5 (range 1-4), 5 pts (63%) had prior venetoclax, 5 pts (63%) had a prior hypomethylating agent, 5 pts (63%) had prior HSCT, and one had prior menin inhibitor. The most common all-grade treatment-related adverse events (TRAEs) in ≥25% of pts were febrile neutropenia (63%), hyperphosphatemia (63%), nausea (63%), and AST/ALT elevation (25%). Grade ≥ 3 TRAEs were febrile neutropenia (63%), decreased platelets count (25%), and decreased neutrophil count (25%). There was 1 dose-limiting toxicity (DLT), grade 4 prolonged thrombocytopenia and neutropenia, at DL0 (resolved after dose hold), prompting enrollment of 3 additional pts, and escalation to DL1 when no additional DLTs were noted (1/6 pts with DLT). Pts at DL1 (N=2) have not completed the DLT period. There were no deaths due to TRAEs. No grade 3 or higher QTc prolongation occurred. Two pts had grade 2 differentiation syndrome (bone pain only), resolved with steroids. One pt ( NUP98r) had asymptomatic leukocytosis at the end of cycle 1 with neutrophilia, monocytosis, decrease in peripheral blasts, decrease in bone marrow blasts from 66% to 8%, and persistence of NUP98r by fluorescence in situ hybridization at 90% indicating differentiation. Seven of 8 pts are response-evaluable, and all 7 attained a morphologic remission (overall response rate of 100%) (Figure 1). Best responses achieved included a complete remission (CR) in 1 pt (including resolution of extramedullary disease), CR with partial hematologic recovery (CRh) in 1 pt, CR with incomplete platelet count recovery (CRp) in 3 pts, partial response (PR) in 1 pt, and 1 pt with morphologic leukemia free state (MLFS). Measurable residual disease was undetectable by flow (sensitivity at 10 -4) in 3 of 7 pts (43%). Three pts transitioned to HSCT following response, 2 are in continued remission, 1 has started maintenance and 1 pt died of transplant complications prior to starting maintenance. Enrollment is ongoing and updated data will be presented. Conclusions: Early results indicate acceptable safety and high efficacy of this combination in R/R myeloid leukemias with either KMT2Ar or NPM1mt or NUP98r. This study is ongoing with plans to establish the recommended phase 2 dose and optimize delivery of this combination.

Combining Menin and MEK Inhibition to Target Poor Prognostic KMT2A-Rearranged RAS Pathway-Mutant Acute Leukemia

Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 ( MLL1/ KMT2A) gene locus with over 90 fusion partners. These fusions are especially prevalent in pediatric acute leukemias. The resulting KMT2A fusion proteins (KMT2A-r) drive leukemogenic gene expression through an interaction with a chromatin complex that includes the scaffold protein menin, giving rise to aggressive acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In infant ALL, frequently characterized by KMT2A-r, the presence of RAS pathway mutations has been associated with a poor prognosis. The prognostic impact of RAS pathway mutations in pediatric KMT2A-r AML is less established. We thus analyzed data from 1605 pediatric patients with AML treated on the Children's Oncology Group (COG) AAML0531 and AAML1031 studies, with 336 of these having KMT2A-r. 51% (170/336) of these patients had mutations in NRAS, KRAS or PTPN11. The combination of KMT2A-r with RAS pathway mutations was associated with worse EFS and OS, HR=1.71 (p<0.001) for EFS and OS multivariable models. Given the prevalence of RAS pathway mutations co-occurring with KMTA-r, and the high-risk leukemia associated with this mutational profile, we sought to identify efficacious targeted drug combinations for this subset of childhood leukemia. Early clinical trial data for the menin inhibitor revumenib (SNDX-5613) demonstrated efficacy in KMT2A-r leukemias. Responding patients, however, typically develop progressive disease with a subset having mutations in MEN1 the gene encoding menin. Effective combination treatments for full efficacy of this targeted therapy are needed. We evaluated the combination of RAS/MAPK targeting using the MEK1/2 inhibitor selumetinib with VTP-50469, a close analog of revumenib, in pediatric leukemias harboring both KMT2A-r and RAS mutations. Treatment of AML and ALL cell lines and cells from an AML patient-derived xenograft (PDX) model in vitro with this drug combination resulted in a synergistic decrease in viability, a G0/G1 cell cycle arrest, and an increase in apoptosis. The combination also resulted in decreased AML colony formation. We next explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. Both selumetinib and VTP-50469 treatment individually reduced phosphorylated ERK levels, but the combination had the most complete inhibitory effect. The combination treatment also resulted in loss of stem-like gene expression signatures, an increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Both MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination. Next, we evaluated the in vivo efficacy of this drug combination in three orthotopic pediatric AML and ALL pediatric PDX models harboring different KMT2A-r and RAS mutations (AML: KMT2A- AF10 with KRAS, KMT2A- PICALM with NRAS, ALL: MLL- AF4 with NRAS). NSG mice engrafted with PDX models via tail vein injection were assigned to four treatment groups: vehicle, chow containing revumenib (0.03%), selumetinib (50 mg/kg/day, 5 days a week), or the combination. After treatment for 18-28 days leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts in 2 models with RAS mutations. In one of the AML models, single agent treatment with revumenib or selumetinib was highly efficacious at eradicating disease at the measured time point. We also found a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting, highlighting on-target activity of the drugs and supporting our in vitro data for a role for MYC in the synergistic effect observed with the combination. In conclusion, new clinical data from a large cohort of COG patients demonstrates that KMT2A-r and co-occurring RAS pathway mutations portend a distinctively worse outcome for children with AML. The findings in our preclinical study suggest a promising, readily translatable targeted treatment for this patient population. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.

Survival Outcomes, Efficacy and Tolerability of a Pediatric-Inspired Regimen (MASPORE) for Philadelphia-Negative Acute Lymphoblastic Leukemia in Young Adults at a Tertiary Hospital in Singapore

Introduction: Recent evidence has emerged to support the use of pediatric-inspired regimen as a standard of care in Adolescents and Young Adults (AYA) patients diagnosed with Philadelphia-negative acute lymphoblastic leukemia (Ph- ALL). The backbone of the pediatric regimen is high cumulative doses of steroids, asparaginase, vincristine and more intensive CNS prophylaxis. Most literature, however, has been in the western population, with few data reported in Asia. MASPORE protocol is a locally designed pediatric-inspired regimen for treatment of ALL in AYA. The protocol uses a 3- or 4-drugs induction regimen (L-asparaginase, Vincristine, Dexamethasone +/- Daunorubicin) based on standard or high-risk disease. PCR-based measurable residual disease (MRD) marker was utilized for disease monitoring and dynamic risk stratification, which led to subsequent intensification of regimen as needed. Methods: In this IRB approved retrospective analysis, patients aged 18-39 years old with Ph- ALL who received MASPORE regimen from January 2009 to December 2021 at the National University Hospital Singapore were identified via our institution database. Treatment outcomes, including measurable residual disease (MRD) monitoring, and toxicity profile were analyzed. Survival outcomes were estimated using Kaplan-Meier method. High risk disease at diagnosis was defined as high white blood cell (>30x10 9/L for B-ALL, >100x10 9/L for T-ALL), hypodiploidy, t(4;11) and other MLL gene rearrangement. Results: 32 patients were identified during the study period; 3 patients were excluded due to incomplete data. The median age was 23 years old (range 18-33 years) with majority being male (86.2%). 16 patients (55.2%) had B-ALL and 13 (44.8%) had T-ALL. High risk disease was present in 7 (43.8%) B-ALL patients and 8 (61.5%) in T-ALL patients. 2 patients in B-ALL group had CNS involvement at diagnosis. The majority (96.5%) achieved complete remission after the induction phase. 1 patient died within first 1 month of treatment due to pancreatitis. 5 patients in high risk group received allogenic stem cell transplantation (alloSCT), whereas 2 patients with standard risk required alloSCT due to relapse and persistent MRD positivity. MRD result was available for 26 patients. In the entire cohort, MRD negativity was achieved in 22 patients (75.9%). Subtype was not associated with achievement of MRD negativity (80% in B-ALL vs 90.9% in T-ALL). High risk patients however had a lower odds of achieving MRD negativity after induction (OR 0.114 95% CI 0.017-0.742, p=0.021). The median follow-up for survivors was 41 months (range 27 to 89 months). Relapse and mortality rate was low at 13.8% each. 5-year OS for patients with B-ALL and T-ALL were 92.3% and 75.2%, respectively (Figure 1a). 5-year OS for patients with standard and high-risk disease were 84.4% and 84.0%, respectively (Figure 1b). Delayed achievement of MRD negativity (i.e. after induction) was not associated with inferior survival outcomes, albeit our sample size was small. In terms of adverse events, Asparaginase-related toxicities included 11 patients (37.9%) with thrombotic events (4 cerebral venous thrombosis and 7 deep vein thrombosis), and 5 patients (17.2%) with grade 2 or more pancreatitis. 7 patients (24.1%) had at least 1 episode of grade 3 or 4 transaminitis. 7 patients (24.1%) had grade 3 or 4 sepsis during the course of treatment. 2 patients developed osteonecrosis of the hip requiring operation and cessation of steroids. Conclusion: Our locally designed MASPORE protocol tailored in accordance to risk stratification demonstrated promising survival outcomes. Similar outcomes observed in standard and high-risk disease suggests the benefit of such an approach. Toxicities were mainly related to L-asparaginase use. Individualized dosing based on drug level monitoring will help to further optimize the treatment intensity with reduced toxicities.

Acquired Venetoclax Resistance in an In Vivo Model of B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Altered Functions of Apoptosis Regulators

Deregulation of cell death pathways such as apoptosis is a hallmark of cancer and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death signaling. Inhibition of anti-apoptotic molecules of BCL-2 family cell death inhibitors has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to BCL-2 resulting in apoptosis induction and has shown clinical activity in hematologic malignancies, but primary and secondary resistance have also been described. In this study, we aimed to identify resistance mechanisms acquired during VEN treatment leading to therapy failure of BCL-2 inhibition. In order to mimic a clinical situation with consecutive treatment cycles, we employed a patient-derived human BCP-ALL xenograft mouse model with subsequent courses of VEN administration to ALL bearing recipients in vivo. First, we estimated VEN-sensitivities in a series of BCP-ALL PDX samples (N=30) by ex vivo titration of individual VEN-EC 50 values and identified a range of heterogeneous VEN-activities. A VEN-sensitive (low VEN-EC50) PDX-ALL sample with a pro-B ALL phenotype carrying a KMT2A:: MLL1 gene fusion was selected and transplanted onto immunodeficient NOD/SCID recipient mice. Upon leukemia manifestation (presence of 5% human ALL cells in peripheral blood), mice were either treated with VEN (100 mg/kg, two times five days) or vehicle control. Recipients were sacrificed upon onset of leukemia-related morbidity and ALL cells isolated from spleens were transplanted onto secondary recipients, which were again treated with VEN or vehicle. ALL cells isolated from secondary recipients were retransferred onto tertiary mice and again exposed to in vivo VEN-treatment providing a total of three subsequent transplantation and in vivo VEN-therapy cycles. Assessing VEN sensitivities of the ALL cells initially transplanted and isolated after VEN-treatment cycles by using an ex vivo cytochrome c release assay showed increasing VEN-resistance. Accordingly, comparison of VEN to control in vivo treated cells showed significantly decreased sensitivities over treatment cycles. Importantly, also the time periods between treatment and leukemia re-occurrence in mice indicating decreasing VEN-sensitivity became shorter with every additional in vivo therapy course. Paired RNA- seq analysis revealed that transcript levels of the pro-apoptotic molecules BAX and BIM were significantly downregulated in VEN-exposed/resistant ALL as compared to vehicle control treated cells. Basal BH3 profiling analysis showed impaired apoptotic priming (significantly lower cytochrome c release after BIM and PUMA peptide exposure) and decreased dependence on anti-apoptotic BCL-2 and MCL-1 indicating a shift from a pro-apoptotic to a pro-survival state. This shift was also reflected in an e x vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC 50 1.5 vs. 2.2 µM, A-1331852 EC 50 9.3 vs. 15.3 µM). In line with the low BAX levels in ALL cells with acquired VEN-resistance, PDX BCP-ALL samples with intrinsic VEN resistance also showed lower BAX protein expression. Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.

MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising treatment for spinal cord injury (SCI), but improving the neurogenic potential of MSCs remains a challenge. Mixed lineage leukemia 1 (MLL1), an H3K4me3 methyltransferases, plays a critical role in regulating lineage-specific gene expression and influences neurogenesis. In this study, we investigated the role and mechanism of MLL1 in the neurogenesis of stem cells from apical papilla (SCAPs). We examined the expression of neural markers, and the nerve repair and regeneration ability of SCAPs using dynamic changes in neuron-like cells, immunofluorescence staining, and a SCI model. We employed a coimmunoprecipitation (Co-IP) assay, real-time RT-PCR, microarray analysis, and chromatin immunoprecipitation (ChIP) assay to investigate the molecular mechanism. The results showed that MLL1 knock-down increased the expression of neural markers, including neurogenic differentiation factor (NeuroD), neural cell adhesion molecule (NCAM), tyrosine hydroxylase (TH), βIII-tubulin and Nestin, and promoted neuron-like cell formation in SCAPs. In vivo, a transplantation experiment showed that depletion of MLL 1 in SCAPs can restore motor function in a rat SCI model. MLL1 can combine with WD repeat domain 5 (WDR5) and WDR5 inhibit the expression of neural markers in SCAPs. MLL1 regulates Hairy and enhancer of split 1 (HES1) expression by directly binds to HES1 promoters via regulating H3K4me3 methylation by interacting with WDR5. Additionally, HES1 enhances the expression of neural markers in SCAPs. Our findings demonstrate that MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. These results provide a potential therapeutic target for promoting the recovery of motor function in SCI patients.

Unlocking the Mysteries of Chromatin Biology through Functional Epigenomics.

In the ever-evolving field of functional genomics, CRISPR-based screening technologies have become pivotal tools for elucidating gene function across various cell types. A recent study by Gilan and colleagues advances this technological frontier by introducing CRISPR-ChIP, a platform designed to investigate the complex dynamics of epigenetic regulation of chromatin. In proof-of-concept experiments, the authors demonstrate the potential of this tool to identify key molecular regulators of two major histone modifications associated with active transcription, H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 79 dimethylation (H3K79me2). They further unveiled a previously unknown functional partitioning of the H3K79-specific methyltransferase DOT1L into an oncogenic complex with MLL-AF9 and a native complex with MLLT10, which cooperatively regulate Mixed Lineage Leukemia fusion protein (MLL-FP) target gene expression. This novel epigenomic approach integrates high-throughput CRISPR screening with ChIP-based direct readout of chromatin modifications in situ, offering a powerful tool to investigate the epigenetic regulatory layers across a diverse spectrum of biological processes and disease states.

B-389 Validation of One-step In-house RT-qPCR Method for Detecting MLL::AF4 Fusion

Abstract Background Rearrangements of mixed lineage leukemia gene (MLL) with 1 of >130 alternative partner genes is a recurrent cytogenetic finding in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is generally associated with a poor prognosis. Among the most common MLL rearrangement is t(4;11)(q21;q23), forming the MLL::AF4 (also known as KMT2A::AFF1) fusion gene. Unique among MLL rearrangements, MLL::AF4 is almost exclusively associated with pro-B cell ALL and is prototypical of infant ALL, where it carries a very poor prognosis. At least 10 different MLL-AF4 fusion transcripts have been found, due to break-points in different introns, some transcripts being more frequent in either adult or infant ALL. Fast and reliable diagnostic tests represent an important tool in clinical laboratories to detect these rearrangements and . In this context, this work aimed to validate a qualitative method to detect the main MLL::AF4 fusion isoforms. Methods We evaluated the performance of the Hs03043614_ft (e10e4, e9e4 and e9e5) and Hs03024412_ft (e11e4) inventoried TaqMan assays (Thermo Fisher Scientific). Total mRNA from negative and positive samples for each fusion was extracted by the TRIzol reagent according to the manufacturer’s instructions. All assays were performed using TaqMan RNA-to-Ct 1-Step kit (Thermo Fisher Scientific). The inventoried ABL1 assay (Thermo Fisher Scientific) was used as endogenous control. Using positive samples, negative samples and controls of each fusion were performed: sequence verification tests, standardization of RNA input, evaluation of reaction efficiency and determination of the detection limit (LOD). The performance of the assays was evaluated with ABL1 control separately from the fusions. Results Our results showed that both assays worked well for the detection of specific fusions. The best RNA input for the reactions was 200 ng. The efficiencies reactions were: a) with ABL1 in separate reactions: 92.6% and 110.0% for Hs03043614_ft and Hs03024412_ft assays, respectively; The literature recommends the limit of efficiency was between 90% and 110%. The LODs with ABL1 in separate reactions were 10−3 for both assays, respectively. There was no unspecific amplification in negative samples. Conclusion The TaqMan assays with RT one-step protocol evaluated are highly sensitive, fast, and reliable tools for detecting the most frequent MLL::AF4 fusion transcripts.

Uncovering NOTCH1 as a Promising Target in the Treatment of MLL-Rearranged Leukemia.

MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings.

Pharmacological inhibition of the mixed lineage leukemia 1-menin interaction aggravates acute kidney injury induced by folic acid and ischemia-reperfusion in mice.

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway.NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.

Effects of low-dose ionizing radiation on genomic instability in interventional radiology workers

Interventional radiologists are chronically exposed to low-dose ionizing radiation (IR), which may represent a health risk. The aim of the present study was to evaluate genomic instability by analyzing chromosomal aberrations, micronuclei, and 53BP1 DNA repair foci in peripheral blood lymphocytes of radiologists. Based on the IAEA guidelines on biodosimetry using dicentrics, the average protracted whole-body dose in radiologists were estimated. Since preleukemic fusion genes (PFG) are the primary events leading to leukemia, we also studied their presence by RT-qPCR and FISH. No significant difference in 53BP1 foci and incidence of PFG (MLL-AF4, MLL-AF9, AML1-ETO, BCR-ABL p190) was found in cells of interventional radiologists in comparison to controls. However, our results showed an increased frequency of micronuclei and various types of chromosomal aberrations including dicentrics in interventional radiologists. The average protracted whole body estimated dose was defined at 452.63 mGy. We also found a significantly higher amplification of the MLL gene segment and increased RNA expression in cells of interventional radiologists in comparison to controls. In conclusion, our results showed that long-term low-dose IR induces genomic instability in interventional radiologists.

Long noncoding RNA Regulating ImMune Escape regulates mixed lineage leukaemia protein-1-H3K4me3-mediated immune escape in oesophageal squamous cell carcinoma.

Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long noncoding RNA (lncRNA) in ESCC immune escape and immunotherapy resistance remains to be elucidated. The tumour-associated macrophage-upregulated lncRNAs and the exosomal lncRNAs highly expressed in ESCC immunotherapy nonresponders were identified by lncRNA sequencing and polymerase chain reaction assays. CRISPR-Cas9 was used to explore the functional roles of the lncRNA. RNA pull-down, MS2-tagged RNA affinity purification (MS2-TRAP) and RNA-binding protein immunoprecipitation (RIP) were performed to identify lncRNA-associated proteins and related mechanisms. In vivo, the humanized PBMC (hu-PBMC) mouse model was established to assess thetherapeutic responses of specific lncRNA inhibitors and their combination with programmed cell death protein 1 (PD-1) monoclonal antibody (mAb). Single-cell sequencing, flow cytometry, and multiplex fluorescent immunohistochemistry were used to analyze immune cells infiltrating the tumour microenvironment. We identified a lncRNA that is involved in tumour immune evasion and immunotherapy resistance. High LINC02096 (RIME) expression in plasma exosomes correlates with a reduced response to PD-1 mAb treatment and poor prognosis. Mechanistically, RIME binds to mixed lineage leukaemia protein-1 (MLL1) and prevents ankyrin repeat and SOCS box containing 2 (ASB2)-mediated MLL1 ubiquitination, improving the stability of MLL1. RIME-MLL1 increases H3K4me3 levels in the promoter regions of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1), constitutively increasing the expression of PD-L1/IDO-1 in tumour cells and inhibiting CD8+ T cells infiltration and activation. RIME depletion in huPBMC-NOG mice significantly represses tumour development and improves the effectiveness of PD-1 mAb treatment by activating T-cell-mediated antitumour immunity. This study reveals that the RIME-MLL1-H3K4me3 axis plays a critical role in tumour immunosuppression. Moreover, RIME appears to be a potential prognostic biomarker for immunotherapy and developing drugs that target RIME may be a new therapeutic strategy that overcomes immunotherapy resistance and benefits patients with ESCC.

Phase separation promotes a highly active oligomeric scaffold of the MLL1 core complex for regulation of histone H3K4 methylation

Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed lineage leukemia (MLL) family of enzymes deposit H3K4 mono-, di-, or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here, we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to the formation of an altered oligomeric scaffold that greatly reduces substrate Km. We posit that phase separation-induced scaffolding of the MLL1 core complex is a potential "switch-like" mechanism for spatiotemporal control of H3K4 methylation through the rapid formation or dissolution of biomolecular condensates within RNA Pol II transcription factories.

Developmental reprogramming of myometrial stem cells by endocrine disruptor linking to risk of uterine fibroids

BackgroundThe stage, when tissues and organs are growing, is very vulnerable to environmental influences, but it’s not clear how exposure during this time causes changes to the epigenome and increases the risk of hormone-related illnesses like uterine fibroids (UFs).MethodsDevelopmental reprogramming of myometrial stem cells (MMSCs), the putative origin from which UFs originate, was investigated in vitro and in the Eker rat model by RNA-seq, ChIP-seq, RRBS, gain/loss of function analysis, and luciferase activity assays.ResultsWhen exposed to the endocrine-disrupting chemical (EDC) diethylstilbestrol during Eker rat development, MMSCs undergo a reprogramming of their estrogen-responsive transcriptome. The reprogrammed genes in MMSCs are known as estrogen-responsive genes (ERGs) and are activated by mixed lineage leukemia protein-1 (MLL1) and DNA hypo-methylation mechanisms. Additionally, we observed a notable elevation in the expression of ERGs in MMSCs from Eker rats exposed to natural steroids after developmental exposure to EDC, thereby augmenting estrogen activity.ConclusionOur studies identify epigenetic mechanisms of MLL1/DNA hypo-methylation-mediated MMSC reprogramming. EDC exposure epigenetically targets MMSCs and leads to persistent changes in the expression of a subset of ERGs, imparting a hormonal imprint on the ERGs, resulting in a “hyper-estrogenic” phenotype, and increasing the hormone-dependent risk of UFs.