Myeloid Differentiation Marker Research Articles

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

Senolytic effects of exercise in human muscles require acute inflammation.

Higher intensity exercise, despite causing more tissue damage, improved aging conditions. We previously observed decreased p16INK4a mRNA in human skeletal muscle after high-intensity interval exercise (HIIE), with no change following equivalent work in moderate-intensity continuous exercise. This raises the question of whether the observed senolytic effect of exercise is mediated by inflammation, an immune response induced by muscle damage. In this study, inflammation was blocked using a multiple dose of ibuprofen (total dose: 1200 mg), a commonly consumed nonsteroidal anti-inflammatory drug (NSAID), in a placebo-controlled, counterbalanced crossover trial. Twelve men aged 20-26 consumed ibuprofen or placebo before and after HIIE at 120% maximum aerobic power. Multiple muscle biopsies were taken for tissue analysis before and after HIIE. p16INK4a+ cells were located surrounding myofibers in muscle tissues. The maximum decrease in p16INK4a mRNA levels within muscle tissues occurred at 3 h post-exercise (-82%, p < 0.01), gradually recovering over the next 3-24 h. A concurrent reduction pattern in CD11b mRNA (-87%, p < 0.01) was also found within the same time frame. Ibuprofen treatment attenuated the post-exercise reduction in both p16INK4a mRNA and CD11b mRNA. The strong correlation (r = 0.88, p < 0.01) between p16INK4a mRNA and CD11b mRNA in muscle tissues suggests a connection between the markers of tissue aging and pro-inflammatory myeloid differentiation. In conclusion, our results suggest that the senolytic effect of high-intensity exercise on human skeletal muscle is mediated by acute inflammation.

Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

Immunophenotyping of blood and bone marrow cells as a way to search for differentiation syndrome risk factors in acute promyelocytic leukemia

Background. Differentiation syndrome (DS) is a potentially fatal complication of therapy for acute promyelocytic leukemia (APL) with an incidence of up to 48 %. To date, no reliable DS risk factors have been found, with the exception of leukocytosis at the APL onset.Aim. To determine the risk factors associated with DS in patients with APL during induction therapy with arsenic trioxide (ATO) and tretinoin (ATRA).Materials and methods. The study included 39 patients with APL, 29 (74.4 %) of them were classified as low-risk according to ELN (European Leukemia Net), 10 (25.6 %) were classified as high-risk. At the disease onset, cytological and molecular (chimeric transcript PML::RARα, FLT3-ITD mutation) bone marrow studies were performed, the expression of 28 differentiation antigens by blood and bone marrow blast cells was determined (markers of early precursors, myeloid and lymphoid differentiation, cell adhesion molecules, chemokine receptors, integrins, selectin), body mass index (BMI) and the leukocytes number dynamics during induction course were assessed. All patients received ATRA and ATO therapy. Patients from the high-risk group at the onset received 1–3 injections of idarubicin (12 mg/m2) and dexamethasone (8–10 mg/m2 2 times a day) to prevent DS until leukocytosis reduced. In cases of DS, dexamethasone was prescribed at a dose of 10 mg/m2 2 times a day; in cases of severe DS, the induction course was interrupted.Results. Of the 39 patients, 12 (30.8 %) were diagnosed with DS: 20 % of high-risk patients (2/10) and 34.5 % of low-risk patients (10/29). There was no statistically significant association of leukocytosis more than 10 × 109 /L at onset, microgranular morphology of blast cells, bcr3-variant PML::RARα, FLT3-ITD mutation with DS. In multivariate analysis, the probability of DS was associated with BMI ≥30 kg/m2 and mean fluorescence intensity of CD38 antigen by blast cells, regardless of risk group. based on the results of the ROC-analysis, the threshold value of mean CD38 fluorescence intensity was set at 25,000 cu, if exceeded, DS is highly likely to develop.Conclusion. the high incidence of DS among low-risk patients is probably due to the lack of prophylactic glucocorticosteroids administration for the development of leukocytosis during ATRA and ATO therapy. BMI ≥30 kg/m2 and mean CD38 fluorescence intensity more than 25,000 cu were identified as statistically significant DS risk factors.

Abstract A013: Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286

Abstract BAF chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer as well as other diseases. FHD-286 is a first-in-class dual inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4) that is being developed for the treatment of advanced hematologic malignancies. We previously reported that exposure of acute myeloid leukemia (AML) cells to pharmacologically relevant concentrations of FHD-286 led to downregulation of leukemic stem cell (LSC) marker CD34, and upregulation of the myeloid maturation marker CD11b. Morphologic features of differentiation were also observed, suggesting that BAF functions to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by reducing their capacity for stem cell renewal. Using single-cell transcriptomic data, we further assessed the impact of FHD-286 on differentiation markers of tumor cells from clinical AML patients. scRNA-seq data was obtained for 26 bone marrow aspirate samples from 11 AML and 2 myelodysplastic syndrome (MDS) patients from the Phase 1 clinical study evaluating FHD-286. Non-tumor cell types (T-cells, erythroid, B cells, megakaryocytes) were identified using published gene markers and removed from the dataset, leaving only tumor cells enriched for either LSC markers (CD34, CD123) or CD11b. We next identified differentially expressed genes between patient LSCs and CD11b+ cells. The top 50 genes associated with each cell group were taken as signature markers of stemness or differentiation, and their mean expression used as phenotypic scores for all patient tumor cells. Clustering analysis of the patient single-cell data showed that cells grouped according to cell type, treatment timepoint, and patient identity. Tumor cells expressing LSC markers CD34 and CD123 clustered separately from those expressing differentiation marker CD11b, and the relative percentage of CD11b+ tumor cells increased on treatment for 8 out of 12 patients dosed at &amp;gt;=5mg, consistent with preclinical AML models. Differential expression analysis revealed additional genes upregulated in either LSCs or CD11b+ cells, which were identified in literature as markers of stemness or myeloid differentiation, respectively. Single-cell LSC and differentiation signature scores showed a strongly statistically significant shift away from LSC identity and toward differentiation for 10 out of 12 patients. Single-cell RNA data from AML patients validates the preclinical observation that FHD-286 treatment induces changes in LSCs resembling differentiation towards a terminal myeloid cell state. This dataset further indicates that nearly all patients undergo some degree of tumor cell differentiation after treatment, suggesting broad on-target activity of FHD-286. Citation Format: GiNell Elliott, Jessica Wan, Kim Horrigan, Mike Collins, Astrid Thomsen, Ashley K Gartin, Ammar Adam, David L Lahr, Gabriel Sandoval, Sameul Agresta, Jessica Piel, Martin Hentemann. Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A013.

Abstract A053: The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML)

Abstract The BRG/BRM associated factor (BAF) complex is a chromatin remodeler critical for maintenance of cell viability in many cancers. This includes acute myeloid leukemia (AML), where BAF maintains the stem-like transcriptional state of blast cells. FHD-286 is a dual inhibitor of the BAF ATPase subunits BRG1 and BRM and is currently being investigated in relapsed or refractory AML and myelodysplastic syndrome. We have shown previously that FHD-286 induces the expression of the myeloid differentiation marker CD11b in preclinical models after treatment with sub-cytoreductive doses at long timepoints. Here we further characterized the differentiation phenotype induced by low-dose FHD-286 in AML cell lines through RNA-seq and a genome-wide CRISPR-Cas9 knockout screen. These datasets and mechanistic follow-up studies suggested AML cell lines treated with FHD-286 can functionally differentiate to gain the ability to produce superoxide anion and perform phagocytosis. We also found that FHD-286 treatment disrupts mRNA splicing and that this is a likely contributor to AML cell growth defects induced by BRG1/BRM inhibition. This study suggests multiple mechanisms by which FHD-286 is able to disrupt the stem-like transcriptional state of AML blasts to cause differentiation and ultimately cell death. Citation Format: Ashley K. Gartin, David N. Mayhew, David L. Lahr, GiNell Elliott, Heena Gandevia, Molly M. Wilson, Richard Centore, Mike Collins, Astrid Thomsen, Jessica Piel, Gabriel J. Sandoval, Martin Hentemann. The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A053.

Abstract B002: Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS

Abstract BRG/Brahma-associated factors (BAF) chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer and other diseases. FHD-286 is a first-in-class dual inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4) that is being developed for the treatment of advanced hematologic malignancies. We previously reported that extended exposure of acute myeloid leukemia (AML) cells to FHD-286 led to upregulation of the myeloid maturation marker CD11b, downregulation of BRG1, and decreased cell proliferation. Morphologic features of differentiation were also observed, suggesting that BAF functions to maintain AML cells in an undifferentiated state and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Biomarkers identified from these nonclinical studies were analyzed in a Phase 1 dose escalation study of FHD-286 monotherapy in patients with relapsed or refractory AML or myelodysplastic syndrome (MDS) (Study FHD-286-C-002). Flow cytometric analysis was performed on serially collected peripheral blood mononuclear cells (PBMCs) from a total of 31 subjects across 4 dose cohorts (2.5, 5, 7.5 and 10 mg once-daily). Peripheral blasts showed time- and dose-dependent upregulation of myeloid maturation markers, including CD11b and CD64, and concomitant decreases in BCL2, CD34, and BRG1. At the highest dose, substantial biomarker modulation was evident beginning at Cycle 1 Day 15 (C1D15), whereas significant modulation was not observed before C2D1 at the lower dose levels. Single cell RNA sequencing of bone marrow aspirates revealed distinct populations of blasts expressing leukemic stem cell (LSC) markers versus those expressing myeloid differentiation markers. In the majority of on-treatment bone marrow samples, blast populations expressing LSC markers were reduced, while populations expressing myeloid differentiation markers were expanded in a generally dose-dependent manner. Evidence of differentiation was also observed by morphological assessment of patient bone marrow samples. Importantly, these effects were observed in subjects with a wide range of mutation profiles, including those considered high risk. In summary, analysis of patient samples from Study FHD-286-C-002 revealed dose-dependent impacts on stemness and maturation markers in peripheral and bone marrow blasts. These results suggest that FHD-286 forces stem-like blasts to mature, possibly reducing their capacity for self-renewal, and leading to a gradual reduction in circulating blasts. Consistent with this, decreases in bone marrow and peripheral blast counts were observed across cohorts. These findings suggest combining FHD-286 with cytoreductive agents may be a promising strategy to target both LSC-like and proliferative blasts, which is supported by nonclinical data showing enhanced antitumor activity with various combination partners. Citation Format: Mike Collins, Astrid Thomsen, GiNell Elliott, Jessica Wan, Kim Horrigan, Laure Delestre, Virginie Penard-Lacronique, Stephane De Botton, Warren Fiskus, Kapil N. Bhalla, Courtney DiNardo, Samuel Agresta, Jessica Piel, Martin Hentemann. Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B002.

Bone Marrow Microenvironment As a Potential Key Regulator of Hematopoietic Cells Stemness in Myelodysplastic Syndromes (MDS)

Background and Rationale. Nuclear Phospholipase C (PLC) beta1 plays a pivotal role in Myelodysplastic Syndromes (MDS) and Azacytidine response [Cocco, L., et al. J Lipid Res. 2015; 56: 1853-60]. Hematopoietic Stem Cells (HSCs) primarily reside within the bone marrow (BM) microenvironment, where mesenchymal stromal cells (MSCs) regulate hematopoiesis through direct interactions with HSCs or cytokine secretion [Blau O, et al. Blood. 2011; 118:5583-92]. The aim of this study was to better elucidate the role of the BM microenvironment in interacting with MDS/AML cells, with particular emphasis on investigating the correlation between PLCbeta1 modulation and the distinctive BM microenvironment response after co-culture, in terms of PLCs gene expression, myeloid differentiation markers and cytokine secretion. Patients and Methods. As it is a very preliminary study, here we analyzed mononuclear cells obtained from 4 higher-risk MDS (RAEB-1, R-IPSS High or Very High, Complex Karyotype), who gave informed consent in accordance with the Declaration of Helsinki. Two patients were treated with Azacytidine (AZA, 75 mg/mq/die for 7 days every 28 days) and one of them reached the fifth cycle with a stable disease. All samples were obtained from the IRCCS-Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy, where clinical evaluations were also conducted. Co-cultures were established with HS-5 MSCs, while Real-Time PCR results were normalized using molecular data obtained from 10 healthy donors (HD). PLCbeta1 overexpression/silencing in vitro model was represented by THP-1 monocytic cells subjected to lentiviral transfection. Both wild-type and PLCbeta1 overexpressing/silencing cells (THP-1 OV/KD) were co-cultured with HS-5 cells for 96 hours. Results. Patients showed distinct basal expression patterns of PLCbeta1: higher in 1/4 MDS and lower in 3/4 MDS, as compared to HD. Irrespective of PLCbeta1 basal expression levels, or response to AZA, in 3/4 patients CD11b increased and CD14 decreased after co-culture. Only one patient, characterized by being a therapy-related MDS, displayed a reduced expression in all myeloid differentiation markers during co-culture. During AZA therapy and after co-culture, one patient showing a stable disease had a significant PLCbeta1 reduction, CD11b increase and CD14 decrease. In THP-1 OV cells co-cultured with HS-5 cells, we observed CD11b reduction, while no significant differences in both PLCs and other myeloid differentiation markers. In contrast, THP-1 KD cells, following co-culture, exhibited a notable increase in CD11b and a decreased CD14 expression. Moreover, THP-1 OV cells alone displayed inhibition of IL-8 secretion, that disappeared after co-culture, in favour of IL-1beta secretion. THP-1 KD cells exhibited suppressed secretion of IL-1alpha, IL-1beta, and IL-8. However, during co-culture, the production of both IL-1alpha and IL-1beta was restored. Conclusions. Our results, that must be confirmed in a larger group of patients, show that the impact of co-culture is not associated with the baseline levels of PLCbeta1, but co-culture recapitulates the PLCbeta1 KD in vitro model and the lack of response to AZA, showing an increase of CD11b and a CD14 reduction. Moreover, the presence of MSCs reversed cytokine secretion, as in co-cultured THP1 KD cells IL-1alpha and IL-1beta were highly secreted again. Considering their multiple roles in both physiological and pathological conditions, ongoing analyses are now further investigating their role in BM microenvironment, also using MDS-MSCs. In addition, we are now exploring the involvement of BM microenvironment in maintaining leukemic cells in a more undifferentiated state and the potential implications of these mechanisms on AZA response/resistance.

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.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p&amp;lt;0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

Retinoic Acid and Ascorbate Remodel the Epigenome of Leukemia Cells to Improve Therapeutic Efficacy By Enhancing TET Activity

Ten-eleven translocation (TET) enzymes are commonly mutated in leukemia, leading to aberrant DNA methylation and impaired hydroxymethylation. All-trans retinoic acid (ATRA) is a vitamin A derivative and cofactor of retinoic acid receptors (RARs) which, in combination with arsenic trioxide, can curatively treat acute promyelocytic leukemia. ATRA has also been shown to upregulate expression of Tet2 and Tet3 in murine embryonic stem cells, and retinol and ascorbic acid (vitamin C) have been shown to enhance pluripotent stem cell reprogramming through increasing hydroxymethylcytosine and promoting DNA demethylation. Our previous work has demonstrated ascorbate can slow disease progression in vivo and, combined with the PARP inhibitor Olaparib, promote cell cycle dysregulation and induce cellular differentiation in acute myeloid leukemia (AML) models both in vitro and in vivo. Here, we show ATRA can induce TET2 and/or TET3 expression in AML cells, and, combined with ascorbate, increase genome-wide oxidized methylcytosine in a TET-dependent manner. Combination treatment with ATRA and ascorbate also induces transcriptional reprogramming, leading to upregulation of DNA repair genes and pathways leading to increased expression of myeloid differentiation markers. Additionally, we observe chromatin remodeling associated with an enrichment for ETS motifs including genes and regions regulated by the transcription factor PU.1, an essential modulator of hematopoietic cell fate. We also show enrichment of retinoic acid receptor A (RARA) at the TET2 locus and at several RAR elements in response to ATRA in AML cells. Using very low dose ascorbate and ATRA, we show that the combination treatment can enhance the efficacy of Olaparib and Venetoclax to alter the cell cycle, promote differentiation, and induce apoptosis of AML cells. These data demonstrate the potential for ATRA and ascorbate to increase TET activity and drive myeloid differentiation and death of AML cells that can be exploited as an adjuvant therapy for the treatment leukemia.

Single-Cell Genomics and Proteomics Reveals Venetoclax-Resistant Monocytic Differentiation of TP53 LOH Clones in TP53 Mutant AML

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of abnormal hematopoietic stem and progenitor cells. TP53 mutations, present in 8 - 30 % of AML, are associated with high risk of relapse and poor prognosis. The mechanisms underlying their persistence are not well understood. Here, we attempt to describe the clonal architecture and immunophenotypes of TP53 mutant ( TP53-mut) clones during therapy. Leveraging advancements in single-cell sequencing and machine learning models, this study integrated single-cell mass cytometry (CyTOF) and single-cell DNA + protein sequencing (seq) profiling of ten paired pre- and post-treatment samples from five TP53-mut AML patients. Methods: Patients 1 and 3 received treatment with CD47AB (Magrolimab), 5'-Azacitidine (Aza), and Venetoclax (Ven); Patient 2 received IMGN632 (CD123-targeting ADC), Aza and Ven. Patient 4 received p97 Inhibitor CB-5339; Patient 5 received CD47 inhibitor (ALX148), Aza, Ven. Consequently, Ven/Aza served as the therapeutic backbone for most patients. For single cell DNA+protein analysis, we sequenced a total of 44,550 cells from these 10 samples. Samples with at least 70% viability were stained with the Total-Seq™-D Human Heme Oncology Cocktail, containing 45 oligo-conjugated antibodies for surface markers relevant to AML. For mutation analysis, we utilized a validated AML custom panel (Morita et al. 2020) covering recurrent mutations in 37 genes. For CyTOF, we multiplexed samples and stained with 50 antibodies characterizing lineage, apoptosis, and signaling pathways. We successfully constructed a machine learning model for integrating both data types using our gradient boosting algorithm established pipelines and were able to predict DNA mutation status in CyTOF data with over 95% accuracy. Results: Analysis of the clonal architecture revealed the presence of TP53 mutations not only in AML blasts but also in lymphocyte subpopulations in all TP53-mut clones from the patients in this study. For example, in patient 1, as a representative case, TP53 mutations were observed in 16.5% of CD4+ T cells, 35.9% of CD8+ T cells, and 19.2% of NK cells. Clonal evolution analysis showed sensitivity of TP53-mut clones with TP53 Loss of heterozygosity ( TP53 mut + LOH) to Ven/Aza and monocytic differentiation in resistant cells, potentially a manifestation of their stem-like nature. Patient 1 displayed three distinct subclones: clone 1 harbored an ETV6 mutation (p.V322S), clone 2, derived from clone 1, acquired additional IDH1 (p.R132L) and TP53 (p.R241G) mutations, clone 3, originating from clone 2 and comprising the highest proportion, exhibited loss of the other TP53 allele (LOH), and decreased from 96.2% to 55.2% post-treatment (Figure 1A). Meanwhile, clone 1 and clone 2 increased from 1.9% to 27.3% and 1.9% to 17.4% respectively. Dissecting the phenotypic changes, clone 3 showed significant pre- and post-treatment shifts in cellular composition. Pre-treatment, it consisted predominantly of 89.7% CD34+ blasts and only 0.2% CD16+ monocytic cells. Post-treatment, CD34+ blasts decreased to 9.3%, while CD16+ monocytic cells increased to 67.8%. CyTOF analysis corroborated our findings (Figure 1B) with trajectory analysis revealing that AML cells had a stemness profile (CD34+CD38- and C-kit+) located at the beginning of the trajectory. Conversely, post-treatment AML cells displayed a loss of stemness and expressed myeloid differentiation markers, including CD33, CD11b, CD14, and CD68. We concluded that Ven-based treatments induce monocytic differentiation in resistant AML cells. CyTOF analysis of the proteomic profiles of resistant cells revealed an increase in Ki67, BCL-XL, and 4EBP1 and a decrease in CD34 and C-KIT, suggesting a potential resistance mechanism involving the upregulation of anti-apoptotic molecules and mTOR pathway. Notably, CD36, a fatty acid transporter, exhibited increased expression in monocytic cells, suggesting an altered metabolism in the survival monocytic cells. Conclusion: This study establishes a genotype-phenotype connection through single-cell proteogenomic profiling of TP53-mutated AML, describing the clonal evolution and immunophenotypic dynamics during treatment while proposing a potential mechanism of resistance.

Acute Myeloid Leukemia Differentiation State and Genotype Influence Anti-Apoptotic Protein Expression, Venetoclax Sensitivity, and Survival in AML

Background: Somatic mutations (mut) and acute myeloid leukemia (AML) differentiation state (phenotype) influence sensitivity and response to treatment. However the interaction between genotype and phenotype with respect to sensitivity or resistance to venetoclax (VEN) based therapy has yet to be fully evaluated. Methods: Samples from patients (pts.) with newly diagnosed (ND) AML enrolled in Beat AML with available bulk RNA sequencing (RNAseq), whole exome sequencing, and ex vivo drug sensitivity testing (N=255) were analyzed for correlation between AML phenotype (defined using RNAseq [van Galen et. al. Cell 2019] and surface immunophenotype via multiparameter flow cytometry [MFC]), genotype, and VEN sensitivity. A monocytic score corresponding to monocytic gene expression and myeloid differentiation for each sample was included as previously defined (Bottomly et. al. Cancer Cell 2022). Clinical outcomes were assessed in an independent retrospective patient cohort (N=97) treated with frontline hypomethylating agents (HMA)+VEN at Oregon Health &amp; Science University. Categorical and continuous variables were analyzed using Fisher's or Wilcoxon rank sum testing. Time to event outcomes utilized the log-rank method with cox multivariable regression modeling. Results: Median patient (pt) age at diagnosis in the surveyed cohort was 61 years (range 20-88). AML phenotype determined using proportional deconvolution of RNAseq data was HSC/progenitor (primitive) in 31% (N=78), mixed/intermediate in 31% (N=79), cDC/monocytic (mature) in 29% (N=73), and unknown in 10% (N=25) of pts. Muts frequent in primitive vs. mature AML samples included transcription factor ( RUNX1, CEBPA, GATA2, GATA1, ETV6, IKZF1; 30% vs. 15%, p=0.050), tumor suppressor ( TP53, PHF6, WT1; 21% vs. 7%, p=0.019), and IDH1/2 (27% vs. 12%, p= 0.027). Active signaling mut ( FLT3-ITD/TKD, K/NRAS, PTPN11, BRAF) were more frequent in mature vs. primitive samples (78% vs. 44%, p &amp;lt; 0.0001), underscoring the correlation between genotype and myeloid differentiation (Fig.A). Expression of anti-apoptotic protein and myeloid differentiation markers measured using RNAseq varied by AML genotype and phenotype and corresponded to VEN resistance. The ratios of BCL2A1 (pearson R=0.62, p &amp;lt; 0.0001), CD11b (R=0.65, p &amp;lt; 0.0001), CD14 (R=0.73, p &amp;lt; 0.0001), and CLEC7A (R=0.72, p &amp;lt; 0.0001) to BCL2 positively correlated with VEN resistance (i.e., increasing area under the curve [AUC]). While IDH1/2 and NPM1 mutated samples demonstrated the highest BCL2 expression and lowest BCL2A1: BCL2 ratio and remained largely sensitive to VEN, myeloid differentiation (in particular monocytic differentiation) increased VEN resistance irrespective of genotype. For instance, classification of a primitive vs. mature phenotype further stratified ex vivo VEN sensitivity in pts with IDH1 (median AUC: 61 vs. 195, p: 0.004), IDH2 (median AUC: 61 vs. 185, p: 0.002), or NPM1 (median AUC: 60 vs. 220, p &amp;lt; 0.0001) mut (Fig.B). Overall survival (OS) following HMA+VEN was evaluated based on the presence or absence of surface immunophenotypic markers measured via MFC that correlated with myeloid differentiation. CD117+ AML (HR: 0.30 95% CI: 0.14 - 0.65, p=0.002) associated with improved OS, while inferior OS was observed with CD11b+ (HR: 2.03 95% CI: 1.07 - 3.86, p=0.031) and CD64+ AML (HR:1.86 95% CI: 0.90 - 3.85, p=0.095). When assessing the combined influence of genotype and immunophenotype on OS in CD11b+ or CD64+ AML, IDH1/2 mut (N=8) numerically improved OS compared to IDH1/2 wild-type cases (N=17) (median 12.3 vs. 5.8 months, p: 0.17). In multivariate analysis adjusted for CD11b or CD64 positivity, age, IDH1/2 mut, and secondary or therapy-related AML, CD11b or CD64 positivity (HR: 1.98 95% CI: 1.08-3.66, p=0.028) retained the strongest impact on OS. Conclusions: Genotype and phenotype both modulate VEN sensitivity in AML. Myeloid differentiation is associated with increased alternative anti-apoptotic protein expression and VEN resistance irrespective of genotype; however certain mutations (i.e., IDH1/2) may retain prognostic significance independent of myeloid differentiation. Given both factors influence VEN sensitivity, prognostic models incorporating AML phenotype and genotype may further improve risk stratification and predict response to VEN based therapy in AML.

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

Preliminary Results from a Phase 1 Dose Escalation Study of FHD-286, a Novel BRG1/BRM (SMARCA4/SMARCA2) Inhibitor, Administered As an Oral Monotherapy in Patients with Advanced Hematologic Malignancies

The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its adenosine triphosphate (ATP)-dependent chromatin remodeling activity, BAF complexes regulate the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. FHD-286 is a first-in-class, orally administered compound that potently and selectively inhibits the ATPase components of the BAF complexes, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). In preclinical models, acute myeloid leukemia (AML) cell lines were highly sensitive to BAF inhibition. FHD-286-C-002 (NCT04891757) is a multicenter, open-label, Phase 1 dose escalation study designed to determine the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose and/or the recommended phase 2 dose(s), as well as to evaluate the preliminary clinical activity of FHD-286, in patients with advanced hematologic malignancies. Patients with relapsed or refractory (R/R) AML or myelodysplastic syndrome (MDS) failing all available standard therapies were eligible to receive FHD-286 doses ranging from 2.5 mg once daily (QD) to 10 mg QD on a continuous dosing regimen. Patients had regularly scheduled assessments to evaluate safety, pharmacokinetics (PK), pharmacodynamics, preliminary clinical activity, and exploratory biomarkers. As of 02 Aug 2022, 40 patients with a median age of 65.5 years (range 25 to 84) with R/R AML (36 patients) or MDS (4 patients) had received at least 1 dose of FHD-286. 67.5% of patients had received ≥3 prior lines of therapy for AML/MDS/other antecedent hematologic disorder, with 22.5% having received ≥5 prior lines. 32.5% of patients had prior hematopoietic stem cell transplant. 65% of patients had adverse risk genetics by 2017 ELN recommendations. 85% of patients experienced a treatment-related adverse event (TRAE) of any grade; the most common (≥20% of patients) were dry mouth (27.5%), increased blood bilirubin (22.5%), and alanine aminotransferase (ALT) increased and rash (20% each). 50% of patients experienced a Grade ≥3 TRAE, the most common of which was increased blood bilirubin (12.5%); stomatitis, ALT increased, differentiation syndrome (DS), and hypocalcemia occurred in 7.5% (each). 2 DLTs were reported: Grade 3 hyperbilirubinemia in 1 patient receiving 5 mg QD and Grade 3 muscular weakness in 1 patient receiving 10 mg QD. Treatment-related DS of any grade was investigator-reported in 4 patients (10%). An independent adjudication committee determined that the rate of suspected DS was 15% (6 patients). 16 patients (15 AML; 1 MDS) had a best overall response of stable disease. Markers of myeloid differentiation with neutrophil recovery and reductions in bone marrow and/or peripheral blasts have been observed in a subset of patients in the study who had a broad range of cytogenetic backgrounds, including patients with enhancer-driven leukemias such as MECOM and KMT2A. Preliminary PK analysis indicates that FHD-286 appears to have a long half-life, at a minimum of &amp;gt;24 hours, and that plasma concentrations of FHD-286 increase with increasing dose. Exploratory flow cytometry assessment of markers of differentiation, hematopoietic stem cell identity, and apoptosis suggested dose-dependent target engagement. Additionally, exploratory sequencing analysis on bone marrow blasts revealed comprehensive impacts on AML-specific expression pathways and stemness genes in response to FHD-286. An exposure-response analysis using the percentage of peripheral blasts showed a trend toward lower blast count with higher FHD-286 area-under-the-curve. Enrollment into the single-agent dose escalation phase of the study is complete. Based on nonclinical, translational, and single-agent clinical data, FHD-286 in combination with decitabine or low-dose cytarabine is being evaluated in the combination dose escalation phase of the study.

IKZF2 Specifically Participates in AML1-ETO Mediated Blockage of Myeloid Differentiation

Objectives:The chromosomal translocation (8;21) is one of the most recurrent cytogenetic aberrations in acute myeloid leukemia (AML). Although most patients achieve complete remission, one third still relapse due to the persistent leukemia stem cells (LSCs). The lack of significant improvements in outcomes, especially in elderly patients ineligible for standard chemotherapy, has spurred further investigation in t(8;21) leukemia. In this study, we investigate the mechanism of AML1-ETO (AE) fusion gene induced differentiation blockage from earlier stages of HSPCs, aiming to identify potential therapeutic targets that can effectively overcome treatment resistance and minimize long-term side effects. Methods:The AML1-ETO conditional knock-in mouse (Aml1 Eto/+; Mx1-Cre) was previously established, hereafter called AE KI. The Cas9-expressing AE KI model, generated by crossing AE KI and Cas9 mice strains, was employed to define the regulatory program of AE fusion gene. For knockout of AE and IKZF2, Kasumi-1 cells were transduced with the single-vector lentiviral system containing sgRNAs for the respective genes, or a nontargeting control. Colony formation and serial bone marrow transplantation assay were performed to evaluate the function of HSPCs. ATAC sequencing, transcriptional profiling (RNA-seq) and Cut&amp;Tag for H3K4me3, H3K27me3, H3K27ac were performed and analyzed comprehensively to characterize the regulatory profile of AE fusion gene. Results:Our previous research demonstrated that induction expression of AE fusion gene in vivo resulted in excessive accumulation of Lin -Sca-1 +c-Kit + (LSK) cells, leading to differentiation blockage from HSCs to HPCs. IKZF2 showed significantly higher expression in LSK cells following PIPC induction of AE expression compared to that of the control group. IKZF2 was highly increased in AML patients harboring AE fusion gene. And the level of IKZF2 was significantly higher in human t(8;21) cell lines Kasumi-1 and SKNO-1 when compared to that of non-AE controls. Depletion of the AE fusion gene led to a dramatic decrease in IKZF2 expression, while constitutive expression of the AE fusion protein in U-937 cells resulted in elevated levels of IKZF2. Loss of IKZF2 resulted in apoptosis in AE-expressing leukemia cells, and led to an increase in the myeloid differentiation markers CD11b and CD14. These findings suggested a functional relevance of IKZF2 in AE-driven AML. In our Cas9-expressing AE KI model, a significant improvement in the peripheral chimerism rate was observed following IKZF2 knockout at various time points. The population of HSCs, mainly the ST-HSCs, was reduced, while the more differentiated MPP and Lin -Sca-1 -c-Kit + (LK) pools, especially the GMP and CMP groups, were expanded. These findings indicated that the differentiation block triggered by AE was partially relieved by IKZF2 knockout. Furthermore, the deletion of IKZF2 had minimal impact on normal myeloid differentiation, highlighting its specific role in AE settings. We further investigated the underlying mechanisms through multi-omics analysis. The induction of AE led to increased accessibility of the myeloid differentiation inhibitory elements, including IKZF2. CUT&amp;Tag data confirmed a significant decrease in H3K27me3 signals at the promoter regions of IKZF2 compared to that of the control group. Regarding the role of IKZF2 in chromatin remodeling, it is likely to interact with AE to form an epigenetic-transcriptional control module. This may expand the signal for differentiation blockage and drive critical leukemogenic gene expression profiles. Conclusions: Our results demonstrate that IKZF2 is a target gene of AML1-ETO crucial for AML1-ETO-induced differentiation blockage from HSCs to HPCs. The epigenetic regulation mechanism may contribute to the AML1-ETO-IKZF2 regulatory axis. And targeting IKZF2 may become a potential therapeutic strategy in human t(8;21) AML especially for the eradication of AML1-ETO-associated LSCs.

Surface immunogenic protein from Streptococcus agalactiae and Fissurella latimarginata hemocyanin are TLR4 ligands and activate MyD88- and TRIF dependent signaling pathways.

The development of vaccine adjuvants is of interest for the management of chronic diseases, cancer, and future pandemics. Therefore, the role of Toll-like receptors (TLRs) in the effects of vaccine adjuvants has been investigated. TLR4 ligand-based adjuvants are the most frequently used adjuvants for human vaccines. Among TLR family members, TLR4 has unique dual signaling capabilities due to the recruitment of two adapter proteins, myeloid differentiation marker 88 (MyD88) and interferon-β adapter inducer containing the toll-interleukin-1 receptor (TIR) domain (TRIF). MyD88-mediated signaling triggers a proinflammatory innate immune response, while TRIF-mediated signaling leads to an adaptive immune response. Most studies have used lipopolysaccharide-based ligands as TLR4 ligand-based adjuvants; however, although protein-based ligands have been proven advantageous as adjuvants, their mechanisms of action, including their ability to undergo structural modifications to achieve optimal immunogenicity, have been explored less thoroughly. In this work, we characterized the effects of two protein-based adjuvants (PBAs) on TLR4 signaling via the recruitment of MyD88 and TRIF. As models of TLR4-PBAs, we used hemocyanin from Fissurella latimarginata (FLH) and a recombinant surface immunogenic protein (rSIP) from Streptococcus agalactiae. We determined that rSIP and FLH are partial TLR4 agonists, and depending on the protein agonist used, TLR4 has a unique bias toward the TRIF or MyD88 pathway. Furthermore, when characterizing gene products with MyD88 and TRIF pathway-dependent expression, differences in TLR4-associated signaling were observed. rSIP and FLH require MyD88 and TRIF to activate nuclear factor kappa beta (NF-κB) and interferon regulatory factor (IRF). However, rSIP and FLH have a specific pattern of interleukin 6 (IL-6) and interferon gamma-induced protein 10 (IP-10) secretion associated with MyD88 and TRIF recruitment. Functionally, rSIP and FLH promote antigen cross-presentation in a manner dependent on TLR4, MyD88 and TRIF signaling. However, FLH activates a specific TRIF-dependent signaling pathway associated with cytokine expression and a pathway dependent on MyD88 and TRIF recruitment for antigen cross-presentation. Finally, this work supports the use of these TLR4-PBAs as clinically useful vaccine adjuvants that selectively activate TRIF- and MyD88-dependent signaling to drive safe innate immune responses and vigorous Th1 adaptive immune responses.

Recombinant Surface Immunogenic Protein from S. agalactiaeand hemocyanin from Fisurella latimarginatapromote cross-presentation dependence on the TLR-4 signaling

Abstract Improved vaccine adjuvants are needed to address infectious diseases, cancer, and future pandemics. Toll-like receptors (TLRs) have been researched as targets of vaccine adjuvants because they modulate the innate immune system and activate adaptive immunity. TLR-based adjuvants induce cross-presentation of exogenous antigens to CD8 T lymphocytes. TLR4 signaling recruits the adapters myeloid differentiation marker 88 (MyD88), which mediates the recycling of endoplasmic reticulum fusion to the phagosome as well as the TIR-domain-containing adapter-inducing interferon-β (TRIF), which is essential for translocation from the endosome to the cytosol. Given the role of TLR4 agonists, most studies have used ligands based on lipopolysaccharides, but these have side effects (especially fever). Here we propose using TLR4 protein-based agonists (TLR-4-PBA), which have multiple advantages as adjuvants: They are naturally biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity. We studied mollusk hemocyanin from Fissurella latimarginata(FLH) and the recombinant surface immunogenic protein from Streptococcus agalactiae(rSIP). These two proteins differ significantly in their structure, and we show Th1 immunomodulatory properties. Using mouse bone marrow-derived dendritic cells pulsed with ovoalbumin (OVA), we then showed that rSIP and FLH promote cross-presentation in OT-I CD8+ T lymphocytes. This effect depends on TLR4 and the recruitment of MyD88 and TRIF. Studies are in progress to evaluate IRF3 and NF-κ B activation.Our results illustrate how these TLR-4-PBAs could act as a clinically useful vaccine adjuvant for innate immunity and Th1 adaptive immune response. FONDECYT 1201600 (MIB); FONDEF ID21I10370 (AEV), Instituto de Salud Pública de Chile Funding (AEV, DD-D); ANID/PhD Nacional program 21210946 (MLS) and 21200880 (DD-D); FONDAP 15130011 (MT, SL).

Plasma Derived Extracellular Vesicles Modulate Monocyte IL-18, IFN-α and IL-10 responses in Adults with HIV-related Cognitive Impairment

Abstract Background: HIV-associated neurocognitive dysfunction persists despite suppressive antiretroviral therapy (ART). Extracellular vesicles (EVs) are emerging modulators of immunological responses. We have previously shown that EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment (CI). Whether these EV changes observed in people with HIV (PWH) influence innate cellular function among those with CI remains unclear. Method: Purified EV samples from 58 HIV-infected individuals on ART with either cognitive impairment (CI, n=48) or normal cognition (NC, n=10) and 8 uninfected controls were isolated using differential centrifugation and co-cultured with PBMCs from a single uninfected donor for 18 hrs. Eight intracellular cytokines and 13 surface chemokines and myeloid differentiation markers were assessed by spectral flow cytometry. Non-parametric statistical T tests were used. Result: Monocytes (mono) showed differential effects on multiple cytokine responses and cell surface receptors after exposure to EVs from HIV-infected individuals with or without CI. Levels of mono IL-18 were significantly lower after incubation with EVs derived from HIV-infected individuals compared to uninfected controls. Based on cognitive status, EVs from HIV-CI group significantly lower % of IL-18 mono responses and lower IFN-α and higher IL-10 responses on a per cell basis when compared to the NC group. Conclusion: These results suggest EVs can modulate mono function irrespective of HIV status. PWH with CI exhibited altered EV driven mono cytokine responses. Our data support interventions that target EVs to reverse or restore mono perturbations and limit neuropathology in PWH on ART. Supported by grants from NIH (R21NS106970 and R01NS117458)

Abstract 2122: The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML

Abstract The BRG1/BRM-associated factors (BAF) complex (also known as mSWI/SNF) is a critical regulator of the chromatin landscape of the genome. By controlling chromatin accessibility, BAF regulates lineage-specific transcriptional programs, including those important for AML blast cell growth and survival. FHD-286 is a highly potent inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4), which demonstrates strong tumor growth inhibition in several AML CDX and PDX models. FHD-286 is being developed for the treatment of relapsed/refractory AML and MDS (see Foghorn website for current status).Investigation of FHD-286 in a broad panel of AML cell lines showed that BAF inhibition affects hematopoietic transcriptional programs important for blast cell self-renewal and identity, agnostic of mutational background. Similarly, FHD-286 demonstrated broad efficacy in ex vivo treatment of AML patient-derived samples from diverse genetic backgrounds, including those with difficult to treat mutational profiles, including mtNPM1, FLT3 ITD, and Inv(3) with EVI1 overexpression. Interestingly, while higher concentrations (≥90 nM) of FHD-286 predominantly induced cytoreduction, lower concentrations (≤30 nM) predominantly led to differentiation-like responses. To investigate this differentiation effect, we performed immunophenotyping of cell lines and primary AML samples following prolonged treatment with FHD-286. Extended treatment (7+ days) with relevant concentrations (5-20 nM) of FHD-286 led to time- and dose-dependent upregulation of the myeloid differentiation marker CD11b, and acquisition of monocyte/metamyelocyte morphology. CD11b+ cells expressed lower levels of the hematopoietic transcription factor PU.1, as well as proliferation and survival proteins Ki67, Myc and BCL2. Additionally, primary patient samples showed a loss of CD34 positivity in blasts and GMP-like cells after 7 days of ex vivo treatment, indicating a decrease in stemness in these populations. Finally, upregulation of CD11b coincided with decreased BRG1 protein, suggesting that immature blasts are characterized by high levels of BRG1. These results suggest that BAF functions to drive transcriptional programs required to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Expanding on these findings, we have also demonstrated synergistic activity with multiple combination partners, including cytarabine and decitabine, in vitro. Elaboration of this in both CDX and PDX in vivo models also shows significant survival benefit in difficult to treat mutational backgrounds. Taken together, these findings suggest that FHD-286 is able to target blast progenitor populations that are heavily BRM/BRG1-dependent, and that combination with standard of care agents can achieve profound, mutationally agnostic antitumor activity in AML. Citation Format: Mike Collins, Astrid Thomsen, Ashley Gartin, Gabriel J. Sandoval, Ammar Adam, Sarah Reilly, Laure Delestre, Virginie Penard-Lacronique, Warren Fiskus, Kapil Bhalla, Stephane de Botton, Sam Agresta, Jessica Piel, Murphy Hentemann. The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2122.