Leukemia Cell Death Research Articles

Synthesis of Alkyl/Aryloxymethyl Derivatives of 1,2,4-Triazole-3-Carboxamides and Their Biological Activities.

Ribavirin and its analogues exhibit an in vitro antiproliferative effect in cancer cells. In this work, we studied the biological activities of a number of alkyl/aryloxymethyl derivatives of ribavirin's aglycon-1,2,4-triazole-3-carboxamide. Alkyl/arylxymethyl derivatives of 1,2,4-triazole-3-carboxamide with substitutions at the fifth or first position of the triazole ring, were synthesized and their antiproliferative and antimicrobial effects were assessed. For both series, the presence of an antiproliferative effect was investigated, and 1-alkyl/aryloxymethyl derivatives were shown an antimicrobial potential against a Gram-positive bacteria Micrococcus luteus and Gram-negative bacterium Pseudomonas aeruginosa. The obtained results showed that the n-decyloxymethyl derivatives induced leukemia cell death at low micromolar concentrations. We confirmed that n-decyloxymethyl derivatives of ribavirin inhibited the cell cycle progression and induced an accumulation of leukemia cells in the subG1-phase. The molecular docking results suggest that alkyl/aryloxymethyl derivatives may act by inhibiting translation initiation, due to interference with eIF4E assembly. The outcome results revealed that active derivatives (1- or 5-n-decyloxymethyl-1,2,4-triazole-3-carboxamides) can be considered as a lead compound for anticancer treatments.

Bioaccessibility of avocado polyhydroxylated fatty alcohols

Avocado-derived polyhydroxylated fatty alcohols (PFAs), such as avocadene and avocadyne, have been recently identified as potent modulators of mitochondrial metabolism which selectively induce leukemia cell death and reverse pathologies associated with diet-induced obesity. However, avocadene and avocadyne bioaccessibility from avocado pulp is not reported; hence, this study aims to investigate if these PFAs are bioaccessible. Dynamic (TNO dynamic intestinal model-1 (TIM-1)) and static in vitro digestion of lyophilized Hass avocado pulp powder shows lipolytic gastrointestinal enzymes led to appreciable bioaccessibility of avocadene (55%) and avocadyne (50%). Furthermore, TIM-1 digestion of a 1:1 ratio of pure avocadene and avocadyne (avocatin B or AvoB) crystals formulated in an oil-in-water microemulsion has on average 15% higher bioaccessibility than the avocado pulp powder demonstrating both dosage forms as potential dietary sources of avocado PFAs. This research provides the impetus for further research on the nutritional significance of dietary long chain fatty alcohols.

Avocado seed extract encapsulated in zein nanoparticles as a functional ingredient

Nano-delivery of natural compounds has gained increasing attention in the food and pharmaceutical ingredients industry. Avocado seed was extracted by a total lipid extraction and the presence of two acetogenin compounds was confirmed by the high-performance liquid chromatography (LC-MS) technique. Then, the avocado seed extract (Avo) was encapsulated into zein nanoparticles by liquid-liquid dispersion and the formation of zein nanoparticles loaded with avocado seed extract (Avo-ZNPs) was confirmed by FTIR analysis. The Avo-ZNPs with various Avo contents showed an average size ranging from 166.9 ± 14.1 to 305.2 ± 16.2 nm with 0.19–0.26 of polydispersity index (PDI), and a surface charge range was −59.5 ± 8.1 to −32.9 ± 0.4 mV, as measured by dynamic light scattering (DLS). The entrapment efficiency (EE) values were up to 82 % and the highest loading capacity (LoC) was 14.8 %. The Avo-ZNPs were found to have significantly higher antioxidant potency compared to the control, as measured by DPPH and FRAP assays. Moreover, the zein nanoparticles were also biocompatible with the acute myeloid leukemia (AML) cell lines, and encapsulated avocado extract induced leukemia cell death. Thus, zein nanoparticles could be a promising delivery system for natural avocado seed extract to be used in the medical and food industries.

Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic and Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Comprehensive characterization of immunogenic cell death in acute myeloid leukemia revealing the association with prognosis and tumor immune microenvironment

BackgroundThis study aimed to explore the clinical significance of immunogenic cell death (ICD) in acute myeloid leukemia (AML) and its relationship with the tumor immune microenvironment characteristics. It also aimed to provide a potential perspective for bridging the pathogenesis of AML and immunological research, and to provide a theoretical basis for precise individualized treatment of AML patients.MethodsFirstly, we identified two subtypes associated with ICD by consensus clustering and explored the biological enrichment pathways, somatic mutations, and tumor microenvironment landscape between the ICD subtypes. Additionally, we developed and validated a prognostic model associated with ICD-related genes. Finally, we conducted a preliminary exploration of the construction of disease regulatory networks and prediction of small molecule drugs based on five signature genes.ResultsDifferentially expressed ICD-related genes can distinguish AML into subgroups with significant differences in clinical characteristics and survival prognosis. The relationship between the ICD- high subgroup and the immune microenvironment was tight, showing significant enrichment in immune-related pathways such as antibody production in the intestinal immune environment, allograft rejection, and Leishmaniasis infection. Additionally, the ICD- high subtype showed significant upregulation in a variety of immune cells such as B_cells, Macrophages_M2, Monocytes, and T_cells_CD4. We constructed a prognostic risk feature based on five signature genes (TNF, CXCR3, CD4, PIK3CA and CALR), and the time-dependent ROC curve confirmed the high accuracy in predicting the clinical outcomes.ConclusionThere is a strong close relationship between the ICD- high subgroup and the immune microenvironment. Immunogenicity-related genes have the potential to be a prognostic biomarker for AML.

Deferasirox Causes Leukaemia Cell Death through Nrf2-Induced Ferroptosis.

Acute lymphoblastic leukaemia (ALL) is the most prevalent cancer in children, and excessive iron buildup resulting from blood transfusions and chemotherapy potentially has a negative impact on treatment outcomes and prognosis in patients with ALL. Therefore, initiating early iron chelation therapy during ALL treatment is a logical approach. Ideally, the selected iron chelator should also possess anti-leukaemia properties. The aim of the present study was to explore the potential impact and underlying mechanism of deferasirox (DFX) in ALL therapy. This study proved that DFX, an iron chelator, is capable of inducing leukaemia cell death through ferroptosis, which is achievable by increasing the expression of acetylated nuclear factor erythroid 2-related factor 2 (NRF2). More specifically, NRF2 acetylation on Lys599 was facilitated by acetyltransferase-p300/CBP. These findings indicate that DFX could serve as a potent adjunctive medication for patients with ALL. Moreover, DFX may offer dual benefits in ALL treatment, functioning as both an iron chelator and NRF2-modulating agent. Further research and clinical trials are necessary to fully elucidate the therapeutic potential of DFX in patients with ALL and incorporate it into treatment protocols.

Abstract 4710: The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism

Abstract Background: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are devastating blood cancers associated with 5-year survival rates of ~50% each in adults. We recently developed a new class of polymeric drugs called PAMDs based on the CXCR4 inhibitor AMD3100 (plerixafor). PAMDs have the potential to be more effective and less toxic therapeutics than the standard of care high-dose chemotherapy. We tested one variant (PAMD-Ch17), and found that unlike AMD3100, PAMD-Ch17 has several novel anti-leukemic effects. To understand the potential mechanism(s) for these new activities, we performed the following study. Methods: To test the role of CXCR4 in PAMD-Ch17’s activities, we generated CXCR4 knockout cells via Crispr/Cas9. We used PrestoBlue, 7-AAD, and Annexin V staining to assay viability and apoptosis. To identify pathways deregulated by PAMD-Ch17, we performed whole transcriptomic sequencing (RNA-Seq). To investigate if PAMD-Ch17 mediates its activities by inducing oxidative stress, we performed SeaHorse assays, and dihydroethidium (DHE), Mitosox, and Bodipy C11 staining. To investigate whether PAMD induces programmed cell death pathways, we used ferrostatin and deferoxamine to inhibit ferroptosis, necrostatin-1 against necroptosis, and Z-VAD-FMK against apoptosis. Results: We found that PAMD-Ch17 induces cell death in a dose dependent manner in human AML and ALL cell lines and, mouse primary leukemia cells, but not healthy mouse bone marrow cells. Surprisingly, PAMD-Ch17 induces equivalent levels of cell death in both wild type and CXCR4 knockout Jurkat cells, indicating that the only known target of the polymer is not required for its anti-leukemic effects. By RNA-Seq, we found altered expression of genes related to mitochondrial function, suggesting that PAMD-Ch17 could be targeting the mitochondria. To test this, we performed SeaHorse assays and found that PAMD-Ch17 induces a significant decrease in mitochondrial respiration. Consistent with this, we found that PAMD-Ch17 induces a significant increase in superoxide production, as well as lipid peroxidation. Using inhibitors of programmed cell death pathways, we found that preventing necroptosis, apoptosis, or ferroptosis caused only subtle effects on PAMD-Ch17 induced cell death, implying that the polymer’s anti-leukemic activity is likely not entirely dependent on these pathways. Conclusions: These results indicate that PAMD-Ch17 induces cell death in leukemia cells but not healthy bone marrow via a CXCR4 independent mechanism. We also found that PAMD-Ch17 induces reactive oxygen species, potentially explaining its selectivity for leukemia cells compared to healthy blood cells. Continued investigation of PAMD-Ch17 will not only further development of this new class of drugs, but also provide much needed insight into leukemia biology. Citation Format: Calvin Lam, Jogdeo Chinmay, Ekta Kapoor, Siyuan Tang, Svetlana Romanova, David Oupicky, Katherine Hyde. The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4710.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Abstract 1687: Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring

Abstract Hematopoiesis relies on an intricate balance between stem-cell self-renewal and terminal maturation of blood cells. Disrupting this balance can lead to the development of deadly blood cancers, like acute myeloid leukemia (AML). Increasing evidence suggests that acquiring hematopoietic stem cell (HSC) gene expression programs in AML confers a particularly poor prognosis and increases risk of relapse with conventional therapy. Acquisition of these features in AML is commonly driven by dysregulation of MECOM, a transcription factor and master regulator of HSC self-renewal. To define the role of MECOM in AMLs, we engineered cell-line models of high-risk AML by endogenously tagging MECOM with an FKBP12F36V degron. These models enable targeted degradation of MECOM which we employed in concert with multi-omic readouts to assess changes in nascent transcription and chromatin accessibility to identify direct transcriptional targets of MECOM. We demonstrate that MECOM degradation results in a rapid increase in chromatin accessibility at MECOM-bound sites and increased expression of cognate genes to establish a pro-differentiation gene program. These findings suggest a previously unappreciated mechanism for MECOM in repressing differentiation-promoting cis-regulatory elements (cisREs) and their cognate target genes in AML. We then employed a pooled CRISPR inhibition (CRISPRi) screen to functionally characterize the role that each cisRE plays in promoting high-risk features in AML. Utilizing CRISPRi to repress MECOM-bound cisREs in concert with dTAG-mediated MECOM degradation, we examined if repression of over 500 individual MECOM-bound cisREs is sufficient to maintain AML progenitors in the absence of MECOM. This functional dissection revealed that the individual repression of three cisREs that regulate CEBPA, GFI1B, and RUNX1 is sufficient to maintain AML progenitor cells in the absence of MECOM. This result implicates MECOM as a direct repressor of known regulators of myeloid differentiation. Given these findings, we hypothesized that small molecule-based recruitment of a transcriptional coactivator to MECOM could rewire its repressive function to activate myeloid differentiation and subsequent leukemia cell death. To test this approach, we treated a MECOM-FKBP12F36V AML degron model with the bifunctional small molecule NICE-01 (AP1867-PEG2-JQ1) to recruit an epigenetic activator (BRD4) to MECOM to functionally rewire this transcriptional network. NICE-01 treatment induced significant differentiation of AML progenitor cells relative to JQ1 treatment alone. Our work highlights the utility of targeted protein degradation to mechanistically interrogate the function of a key driver of high-risk AMLs and suggests the potential for small molecule-rewiring of stem cell gene regulatory networks to confer therapeutic benefit. Citation Format: Travis J. Fleming, Michael Gundry, Richard Voit, Mateusz Antoszewski, William J. Gibson, Ananthan Sadagopan, Vijay G. Sankaran. Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1687.

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2–3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Targeting the TNF/IAP pathway synergizes with anti-CD3 immunotherapy in T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Current treatments, based on intensive chemotherapy regimens provide overall survival rates of ∼85% in children and <50% in adults, calling the search of new therapeutic options. We previously reported that targeting the T-cell receptor (TCR) in T-ALL with anti-CD3 (αCD3) monoclonal antibodies (mAbs) enforces a molecular program akin to thymic negative selection, a major developmental checkpoint in normal T-cell development; induces leukemic cell death; and impairs leukemia progression to ultimately improve host survival. However, αCD3 monotherapy resulted in relapse. To find out actionable targets able to re-enforce leukemic cells’ vulnerability to αCD3 mAbs, including the clinically relevant teplizumab, we identified the molecular program induced by αCD3 mAbs in patient-derived xenografts derived from T-ALL cases. Using large-scale transcriptomic analysis, we found prominent expression of tumor necrosis factor α (TNFα), lymphotoxin α (LTα), and multiple components of the “TNFα via NF-κB signaling” pathway in anti-CD3–treated T-ALL. We show in vivo that etanercept, a sink for TNFα/LTα, enhances αCD3 antileukemic properties, indicating that TNF/TNF receptor (TNFR) survival pathways interferes with TCR-induced leukemic cell death. However, suppression of TNF-mediated survival and switch to TNFR-mediated cell death through inhibition of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) with the second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant synergizes with αCD3 to impair leukemia expansion in a receptor-interacting serine/threonine-protein kinase 1–dependent manner and improve mice survival. Thus, our results advocate the use of either TNFα/LTα inhibitors, or birinapant/other SMAC mimetics to improve anti-CD3 immunotherapy in T-ALL.

Hellebrigenin triggers death of promyelocytic leukemia cells by non-genotoxic ways

Bufadienolides are digitalis-like aglycones mainly found in skin secretions of toads. Among their biological properties, the mechanisms of antiproliferative action on tumor cells remain unclear for many compounds, including against leukemia cells. Herein, it was evaluated the mechanisms involved in the antiproliferative and genotoxic actions of hellebrigenin on tumor cell lines and in silico capacity to inhibit the human topoisomerase IIa enzyme. Firstly, its cytotoxic action was investigated by colorimetric assays in human tumor and peripheral blood mononuclear cells (PBMC). Next, biochemical and morphological studies were detailed by light microscopy (trypan blue dye exclusion), immunocytochemistry (BrdU uptake), flow cytometry and DNA/chromosomal damages (Cometa and aberrations). Finally, computational modelling was used to search for topoisomerase inhibition. Hellebrigenin reduced proliferation, BrdU incorporation, viability, and membrane integrity of HL-60 leukemia cells. Additionally, it increased G2/M arrest, internucleosomal DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization in a concentration-dependent manner. In contrast to doxorubicin, hellebrigenin did not cause DNA strand breaks in HL-60 cell line and lymphocytes, and it interacts with ATPase domain residues of human topoisomerase IIa, generating a complex of hydrophobic and van der Waals interactions and hydrogen bonds. So, hellebrigenin presented potent anti-leukemic activity at concentrations as low as 0.06 μM, a value comparable to the clinical anticancer agent doxorubicin, and caused biochemical changes suggestive of apoptosis without genotoxic/clastogenic-related action, but it probably triggers catalytic inhibition of topoisomerase II. These findings also emphasize toad steroid toxins as promising lead antineoplasic compounds with relatively low cytotoxic action on human normal cells.

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

Active Hexose-Correlated Compound Shows Direct and Indirect Effects against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

Hexamethylene amiloride synergizes with venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia

Tumors maintain an alkaline intracellular environment to enable rapid growth. The proton exporter NHE1 participates in maintenance of this pH gradient. However, whether targeting NHE1 could inhibit the growth of tumor cells remains unknown. Here, we report that the NHE1 inhibitor Hexamethylene amiloride (HA) efficiently suppresses the growth of AML cell lines. Moreover, HA combined with venetoclax synergized to efficiently inhibit the growth of AML cells. Interestingly, lysosomes are the main contributors to the synergism of HA and venetoclax in inhibiting AML cells. Most importantly, the combination of HA and venetoclax also had prominent anti-leukemia effects in both xenograft models and bone marrow samples from AML patients. In summary, our results provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML invitro and invivo.

The RORɣ/SREBP2 pathway is a master regulator of cholesterol metabolism and serves as potential therapeutic target in t(4;11) leukemia

Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon RORγ inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the RORγ inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia.

Serine Phosphorylation Marks Proteins for Degradation By the Mitochondrial Serine Protease Complex, Clpxp

ClpXP, a serine protease located in the mitochondrial matrix, regulates mitochondrial proteostasis by degrading damaged or aggregated proteins, including respiratory chain subunits. ClpXP is a bipartite protein complex comprised of the ClpX regulatory particle that caps each end of the ClpP degradation chamber. ClpX recognizes substrates, unfolds them, and feeds them into ClpP for proteolysis. Genetic or chemical inhibition of ClpXP leads to impaired oxidative phosphorylation function and leukemic cell death in vitro and in vivo. Importantly, however, the degradation marker of human ClpXP remains elusive. Since the bacterial homologue of ClpXP recognizes phosphorylated arginine (pArg)-tagged protein for degradation, we hypothesized that human ClpXP might also recognize phosphorylated amino acids as degrons. We first investigated whether phosphorylation influences substrate degradation by ClpXP using a model substrate, α-casein. Recombinant human ClpXP preferentially degraded phosphorylated α-casein compared to dephosphorylated α-casein. To further elucidate the effects of phosphorylation, we screened a panel of phosphorylated amino acids for their ability to impede ClpXP-mediated degradation of FITC-tagged α-casein. Phosphorylated serine (pSer) and phosphorylated threonine (pThr) free amino acids, or short peptides containing pSer or pThr, inhibited α-casein degradation by ClpXP in a dose-dependent manner. In contrast, phosphorylated pArg, and phosphorylated tyrosine (pTyr), as well as free unmodified Ser or Thr, did not impact ClpXP protease activity. Next, we applied a thermal shift assay to measure the binding capabilities of pSer and pThr to ClpX. We discovered that these phosphorylated amino acids, whether free or incorporated into short peptides, successfully associated with ClpX. In contrast, their dephosphorylated counterparts did not exhibit the same binding activity. Notably, pSer did not affect enzyme activity of the related LonP1 mitochondrial matrix serine protease, thereby demonstrating the specificity of pSer for ClpXP. Extending our studies to intact cells, we analyzed a proteomic dataset of post-translational modifications in Jurkat cells with and without treatment with bortezomib (1 µM), a proteasome inhibitor that inhibits ClpXP at this concentration. Notably, a global enrichment of phosphorylation was observed for mitochondrial proteins upon bortezomib treatment. We overlaid observed phosphorylated proteins with the ClpXP interactome as determined by BioID, and identified 9 phosphorylated mitochondrial proteins that also interact with ClpXP. Of these proteins, respiratory chain II complex subunit, SDHA, was the top hit, where serine phosphorylation was increased over 4 folds. Next we determined how depletion of ClpXP affected levels of phosphorylated substrate in intact cells. Knockdown of ClpX or ClpP in OCI-AML2 cells increased levels of serine phosphorylated SDHA (pSer-SDHA). We then added recombinant ClpXP protein to mitochondrial lysates and observed selective degradation of pSer-SDHA. To determine if pSer-SDHA are damaged proteins, we partitioned mitochondria into soluble and insoluble fractions using digitonin. pSer-SDHA was enriched in the detergent-insoluble fraction. In addition, we induced mitochondrial proteolytic stress by treating OCI-AML2 cells with antimycin to increase mitochondrial ROS or through heat shock by culturing cells at 42 oC. Both antimycin and heat shock increased pSer-SDHA in the detergent-insoluble fraction, further supporting pSer marking damaged proteins. Finally, we developed small molecule pSer mimics. We showed that these compounds bound ClpX in thermal shift assays. In addition, these compounds inhibited ClpXP protease activity in cell-free enzymatic assays. In summary, ClpXP recognizes serine phosphorylation as a degradation marker for damaged mitochondrial proteins. Small molecules that inhibit ClpX could be leads for novel anti-cancer agents.

KDM6A Modulates Anti-Tumor Immune Response By Integrating Immunogenic Cell Death in Human Acute Myeloid Leukemia

Identifying tumor cell-epigenetic regulation of immune activation in acute myeloid leukemia (AML) would be instrumental in developing next-generation of targeted therapy. AML cells have low mutational burden; inefficient ‘cross-presentation’ through conventional dendritic cells (DC) and immune activation are the major challenges for AML immunomodulation. ‘Immunogenic cell death’ (ICD) represents changes in the composition of the tumor cell surface as well as the release of ‘damage associated molecular patterns’ (DAMPs) in the tumor microenvironment that stimulates the dysfunctional immune system. DAMPs in turn operate on a series of receptors expressed by DCs and natural killer (NK) cells and contribute to long-lasting, protective anti-tumor immunity. For example, plasma membrane exposure of endoplasmic-reticulum (ER)-translocated calreticulin (ecto-CRT), which functions as an ‘eat me’ signal, is an important hallmark of ICD. Previous study suggested that calreticulin (CALR) promotes immunity and type I interferon-dependent survival in mice with AML (Fucikova, Blood 2016; Chen, Oncoimmunology 2017). In addition, ecto-CRT on malignant blasts correlated with improved NK cell-mediated cytotoxicity in AML patients with superior overall survival (Truxova, Haematologica 2020). Ecto-CRT has also been identified as an endogenous sterile ligand for NK cells (Sen Santara, Nature 2023). Nevertheless, molecular regulation of ICD in the context of epigenetic derangements and inflammation and its implication in AML immunotherapeutic modulation is poorly understood. KDM6A is a histone 3 lysine 27 demethylase that plays tumor suppressor function in AML (Gozdecka, Nat Genet 2018; Sera, Blood 2021). KDM6A escapes X-chromosome inactivation, and loss-of-function deletion or point mutations associate with resistance to ‘3+7‘ chemotherapy (Stief, Leukemia 2020). We have identified that in comparison to control AML cells, deficiency of KDM6A ( KDM6A-kd) associated with daunorubicin/etoposide-induced ICD, which was manifested by significant increase (2-3 fold , P&amp;lt;0.05) in ecto-CRT, plasma membrane exposure of pE1F2α, HSP70 and HSP90 with extracellular release of HMGB1 and ATP. Similarly, pharmacological inhibition of JmjC catalytic function using GSK-J4 sensitized human AML lines as well as primary AML patient bone marrow-derived CD34 + hematopoietic stem/progenitor cells to ICD. Induction in ICD was accompanied by an elevated type I interferon production that was functionally active in promoting interferon regulated gene expression. Interestingly, treatment with acute recombinant IFN-β caused an increase in activated STAT1 and inflammatory gene expression in KDM6A-kd AML cells compared to control AML. Corroborating these results, gene set enrichment analysis revealed a significant enrichment (NES=1.708, P=0.007) of upregulated type I interferon signatures (n=40) in Kdm6a -/- primary leukemia cells. Re-analysis of ChIP-seq results indicated 1647 Kdm6a occupied genes, which were upregulated upon Kdm6a loss. Gene ontology (GO) analysis of the 1647 genes further suggested an enrichment ( P&amp;lt;0.001) of type I interferon-responsive GO terms, indicating Kdm6a role in interferon signaling. Ecto-CRT level relies on premortem ER stress. In OHSU AML, KDM6A expression correlates with ER stress/unfolded protein response. Mechanistically, quantitative-ChIP and promoter-reporter assays indicated that KDM6A-kd de-repressed ICD induced expression of ATF4 and HSPA5 through chromatin and transcriptional remodeling. In agreement with these findings, AML blasts-healthy immune cells ex vivo co-culture experiments revealed that increase in ICD in KDM6A-kd AML was associated with activation (~2-foldincrease inCD80 +CD86 +, P&amp;lt;0.05) and phagocytic function of DCs. Loss of KDM6A was shown to bestow ‘BRCAness’ (Boila, Leukemia 2023), and PARP inhibition itself can cause ICD and augment tumor immunity. We observed that there was a significant induction in ICD and expression of NKG2D ligands ULBP1, ULBP5 in KDM6A-kd AML cells treated with olaparib. In addition, olaparib and GSK-J4 treatment showed additive effects in ICD and immune activation using primary AML CD34 + cells. Together, we illustrate that KDM6A regulates ICD through epigenetic mechanisms, while KDM6A deficient AML subtypes could be sensitized with immunomodulatory targeted therapy.