Acute Myelogenous Leukemia Cells Research Articles

Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager.

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.

Abstract LB157: Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies

Abstract E1A-binding protein P300 (p300) and its paralog CREB binding protein (CBP) are key acetyl transferases (HAT) and transcriptional co-factors. p300/CBP bromodomain (BRD) inhibition reduces acetylation of H3K27 at enhancers, leading to suppression of enhancer-driven gene expression, such as MYC and IRF4 that are critical for the initiation, progression, and chemoresistance of hematological malignancies. Thus, p300/CBP inhibition is a highly potential therapeutic strategy for the treatment of hematological malignancies, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL) and leukemia. We have discovered HP537 that competitively inhibits an acetylated peptide substrate to bind BRD of p300/CBP with IC50 < 5 nM. HP537 does not inhibit the enzymatic activity of HAT domain of p300/CBP and HAT1, nor has binding affinity for other BRD containing proteins including BRD4 and BRD9. HP537 inhibits proliferation of a wide range of cell lines derived from MM, leukemia and lymphoma with IC50 values ranged from 0.01 μM to 0.4 μM. Protein expression levels of acetylated H3K27, c-Myc and IRF4 are significantly decreased by HP537 in MM, NHL and acute myelogenous leukemia (AML) cell lines in a dose- and time-dependent manner. HP537 robustly and dose-dependently inhibits tumor growth in OPM-2 (MM), MOLM-16 (AML), and KARPAS-422 (NHL) cell line derived tumor xenograft (CDX) mouse models. Down-regulation of c-Myc and IRF4 are also observed in the tumor samples. In conclusion, HP537 is a novel, potent and selective inhibitor of p300/CBP BRD. The pre-clinical data supports the clinical development of HP537 for the treatment of hematological malignancies. A FIH study of HP537 in cancer patients is planned to start in 2024. HP537 structure will not be disclosed. Citation Format: Jing Li, Zhilin Tu, Jian Peng, Lei Fan, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB157.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Therapeutic Targeting of TIM-4-L with Engineered T Cells for Acute Myeloid Leukemia.

Disruption of lipid bilayer asymmetry is a common feature observed in cancer cells and offers novel routes for therapeutic targeting. We used the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples and evaluated the anti-leukemic activity of TIM-4-L-directed T-cell therapy in preclinical AML models. We performed FACS analysis on 33 primary AML bone marrow specimens and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. Using Kasumi-1 and MV-4-11 AML cell lines, we further tested the anti-leukemic effects of TIM-4-L-directed engineered T cells in vitro and in vivo. We found that 86% of untreated AML blasts displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1 displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. TIM-4-L dysregulation was also stably present in AML cell lines. To evaluate the potential of targeting upregulated TIM-4-L with adoptive T-cell therapy, we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines with a range of endogenous TIM-4-L expression levels both in vitro and in vivo. These results highlight TIM-4-L as a highly prevalent target on AML across a range of genetic classifications and novel target for T-cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.

Biochips to measure ultraviolet light absorption by leukemia cells

During the treatment of leukemia, side effects create a great burden for patients. Therefore, new therapies to replace conventional chemotherapy and hematopoietic stem cell transplantation are highly desirable. In a previous study, I found that UV irradiation at 365 nm induced apoptosis in 98% of leukemia (Jurkat T) cells. To determine the most suitable wavelength for such treatments, it is necessary to measure the UV light absorption of cancer cells. In this study, a biochip was fabricated and evaluated for this purpose. Three types of leukemic cells (human acute myelogenous leukemia cells, human acute lymphoblastic leukemia cells, and human chronic myelogenous leukemia cells) were injected into the biochip, and light absorption was measured, revealing that individual leukemic cells have different light absorption characteristics. This variation is important in order to develop an implant care device to treat leukemia more effectively.

Discovery of novel selective phosphodiesterase‑1 inhibitors for the treatment of acute myelogenous leukemia

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. PDE1 (Phosphodiesterase 1) is a subfamily of the PDE super-enzyme families that can hydrolyze the second messengers cAMP and cGMP simultaneously. Previous research has shown that suppressing the gene expression of PDE1 can trigger apoptosis of human leukemia cells. However, no selective PDE1 inhibitors have been used to explore whether PDE1 is a potential target for treating AML. Based on our previously reported PDE9/PDE1 dual inhibitor 11a, a series of novel pyrazolopyrimidinone derivatives were designed in this study. The lead compound 6c showed an IC50 of 7.5 nM against PDE1, excellent selectivity over other PDEs and good metabolic stability. In AML cells, compound 6c significantly inhibited the proliferation and induced apoptosis. Further experiments indicated that the apoptosis induced by 6c was through a mitochondria-dependent pathway by decreasing the ratio of Bcl-2/Bax and increasing the cleavage of caspase-3, 7, 9, and PARP. All these results suggested that PDE1 might be a novel target for AML.

Estimation of Serum TLR-9,TNF-α, and IL-6 Levels in the Iraqi Patients Diagnosed as Acute Myelogenous Leukemia

Acute myelogenous leukemia (AML) is a varied group of biological and clinical diseases. It is distinguished by the fast proliferation of aberrant cells in the bone marrow, which interferes with normal blood cell formation. Several studies demonstrated that cytokines released by leukemic cells in an autocrine or paracrine manner influence the proliferation of AML cells. Our study aimed to assess serum levels of TLR-9, TNF-α and, IL-6 in patients of AML. The study was done via the ELISA Technique. The results show a highly significant difference in all parameters in Patients and control. The correlation between immunological parameters using the Pearson correlation coefficient for patients showed that there is a weak correlation between TLR-9 and TNF- α, IL-6 with P-values of 0.47 and 0.23 respectively. This may indicate the complex environment of inflammation. However, there was a strong correlation between TNF- α and IL-6 with a P-value (0.001). This indicates that the secretion of these cytokines was high in patients. All immunological parameters of patients were evaluated using the receiver operating characteristic (ROC) curve to demonstrate that any one of them is a useful tool for identifying and tracking inflammation. The ROC curve's findings revealed that all parameters have good sensitivity and specificity for detecting inflammation and disease activity in AML patients. According to the current study, AML was related to higher levels of TLR-9, TNF-α, and, IL-6 may be affected the prognosis of the disease, increase the risk of the disease progression, and may be used as a biomarker to target AML cells.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

High Expression Level of MMP7 and Mir-489-3p/MMP7 Axis Associates with Venetoclax Resistance in Acute Myelogenous Leukemia

The BCL2 inhibitor venetoclax used to treat acute myelogenous leukemia (AML). Although this has been a promising therapeutic option for patients with AML, many of these patients develop resistance and relapsed disease. Matrix metalloproteinases (MMPs) were demonstrated to have important implications in the progression and invasiveness of many malignant disorders. In contrast, the biological significance of these molecules in AML is not clear. We determined the levels of MMP-7 in the bone marrow of 76 patients with AML. 70 bone marrow donors served as normal controls. The level of MMP-7 was significantly higher in the AML than in the normal controls. MMP-7 levels were significantly lower in the AML patients who achieved a complete remission (CR) than in those who did not. Through in vitro studies, we observed that MMP7 levels were elevated in AML cell lines (U937, THP-1) and in Venetoclax-resistant AML cell lines (U937R, THP-1R) and that downregulation of MMP7 could enhance Venetoclax efficacy in U937R and THP-1R cells. Our in vitro and in vivo investigations show that miR-489-3p was downregulated in Venetoclax-resistant AML cell lines and promoting level of miR-489-3p expression can directly suppressing MMP-7 expression. Pharmacological inhibition of miR-489-3p markedly reversed the effects of Venetoclax-resistant upon AML proliferation and metastasis. Thus, our study suggests that MMP-7 is an essential factor in AML, and regulator the miR-489-3p/MMP7 axis may be a novel treatment strategy for overcoming Venetoclax resistance in AML.

Hematopoietic stem cells suppress proliferation and enhance differentiation of leukemia cells through regulating apoptotic and inflammatory genes.

Stem cells are known to play an important role in tumor treatment and many of them have shown tumor-suppressing ability in different cancers; however, whether hematopoietic stem cells (HSCs) have growth-inhibiting effects on leukemia cells has not been fully evaluated. Herein, we aimed to demonstrate the growth-restraining function of HSCs in acute leukemia treatment. Cell fusion experiment was conducted by PEG-1500. The viability, proliferation, apoptosis and differentiation of leukemia cells were evaluated by cell counting, CCK-8 and flow cytometry analysis. The morphological changes were imaged using a fluorescence microscope. The expression of genes was detected by quantitative reverse transcription PCR (qRT-PCR). We observed that HSCs and their lytic extracts had the capability to suppress leukemia cells proliferation, promote apoptosis and especially induce acute myelogenous leukemia (AML) cells differentiation, which might have an effect on differentiation therapy to leukemia especially AML treatment. The expression levels of Bcl-2, Survivin decreased and Bax increased following HSCs extracts treatment. Furthermore, the expression of inflammatory cytokines also changed in AML cells which might have to do with the mechanism of HSCs/extracts suppressing effect. HSCs and their extracts can suppress the proliferation of leukemia cells and enhance the differentiation of AML cells and using the extracts of HSCs might be a probable therapeutic option for acute leukemia.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients.

Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Our photodynamic therapy (PDT) approach has been shown to efficiently destroy leukemia cells from tumor-infiltration mimicking models (TIMs), indicating the feasibility of this technique to purge OT samples. Autotransplantation of cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women who require immediate cancer treatment. Up until now, more than 200 live births have already been reported after OT cryopreservation and transplantation. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age and in 2020, the estimated number of new leukemia cases was higher than 33000 in girls between 0 and 19 years old. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient leading to leukemia recurrence. To safely transplant the OT from leukemia patients and restore their fertility, our goal was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging of acute myelogenous leukemia cells from OT fragments (n = 4). Moreover, to ensure that such treatments are not harmful to follicle survival and development so they can be deemed a potential fertility restoration alternative, the effect of the ORN-based PDT purging procedure on follicles was assessed after xenografting the photodynamic-treated OT in SCID mice (n = 5). The work was carried out between September 2020 and April 2022 at the Catholic University of Louvain. After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 cells from ex vivo TIMs prepared by microinjection of a cancer cell suspension into OT fragments. The purging efficiency was analyzed by droplet digital polymerase chain reaction and immunohistochemical analyses. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival and development, and tissue quality in terms of fibrotic areas and vascularization after 7-day xenotransplantation to immunodeficient mice. The ex vivo purging of TIMs demonstrated that our PDT strategy could selectively eradicate the malignant cells from tissue fragments without affecting OT normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and PDT-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, our findings showed that the control and PDT-treated OT could be equally vascularized (7.65 ± 1.45% and 9.89 ± 2.21%, respectively). Similarly, the proportions of fibrotic area did not differ between the control (15.96 ± 5.94%) and PDT-treated groups (13.32 ± 3.05%). N/A. This study did not use OT fragments from leukemia patients, but TIMs created after injection of HL60 cells into OT from healthy patients. Therefore, while the results are promising, whether our PDT approach will be equally successful in eliminating malignant cells from leukemia patients remains to be assessed. Our results showed that the purging procedure causes no significant impairment effect on follicle development and tissue quality, suggesting that our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of OT, allowing safe transplantation in cancer survivors. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A.); Fondation Louvain (awarded to C.A.A.; a Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and a Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer); and Foundation Against Cancer (grant number 2018-042 awarded to A.C.). The authors declare no competing interests.

Mechanisms of response and resistance to combined decitabine and ipilimumab for advanced myeloid disease

AbstractThe challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Dual mTORC1/2 Inhibition Synergistically Enhances AML Cell Death in Combination with the BCL2 Antagonist Venetoclax.

Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.

The RNA m6A Reader YTHDF1 Is Required for Acute Myeloid Leukemia Progression.

The m6A reader YTHDF1 is required for progression of acute myelogenous leukemia and can be targeted with the FDA-approved drug tegaserod to suppress leukemia growth.

Abstract IA020: Therapeutic targeting of MLL complexes in cancer

Abstract Inappropriate expression of genes such as the homeotic (HOX) genes is found in up to 40% of cases of Acute Myelogenous Leukemia (AML). Two well-defined genetic subsets, those with MLL-rearrangements and those with NPM1 mutations, are known to possess high-level expression of HOX and MEIS1 genes and have been shown to be dependent on continued expression of these genes. Recent studies have defined the chromatin-bound protein complexes that maintain this aberrant gene expression. Furthermore, multiple groups including ours have now developed small molecule inhibitors of chromatin-associated complexes. Some of these small molecules, including those targeting DOT1L, EZH2, and Bromodomains are at various stages of clinical development. Another approach to target chromatin associated complexes is via disruption of protein-protein interactions. One such protein-protein interaction that is being targeted is the interaction between the histone methyltransferase MLL1 and Menin, an approach that has also been shown to reverse leukemogenic HOX/MEIS1 gene expression. We have developed novel, potent and orally bioavailable MLL1-Menin inhibitors and have brought one of these small molecules to clinical assessment. Inhibition of the MLL1-Menin interaction in MLL-rearranged and NPM1 mutant AML cells leads to loss of Menin from chromatin, destabilization of the Menin protein and specific changes in chromatin state that include loss of the MLL1 complex at select loci. This results in selective reversal of leukemogenic gene expression, changes in occupancy of multiple other chromatin complexes, rapid cellular differentiation and cell death. Also, we have recently found this interaction to be critical in subtypes of Sarcoma opening up novel potential therapeutic opportunities. These molecules have remarkable preclinical activity in human disease models including patient derived xenograft (PDX) models, show minimal toxicity and are now in phase I/II clinical trials. Inhibition of protein-protein interactions represents a potentially effective approach to destabilize chromatin associated complexes for cancer therapy. Citation Format: Scott A. Armstrong. Therapeutic targeting of MLL complexes in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA020.

Novel Small Molecule and Peptide Inhibitors of CREB in Leukemia Cells

Background: Acute Lymphoblastic Leukemia (ALL) and Acute Myelogenous Leukemia (AML) account for nearly 45% of all childhood cancer. Current therapy for acute leukemia results in significant morbidity and late complications in children. Therefore, more effective and less toxic therapies to treat leukemia are needed. Previous work in our laboratory has identified CREB (cAMP Response Element Binding Protein), a transcription factor that promotes cell proliferation and survival in acute leukemia, is overexpressed in primary ALL and AML cells and is a potential target for therapy. We have investigated small molecules and peptides to inhibit the critical interaction between the phosphorylated KID (Kinase-Inducible) domain of CREB and the KIX (KID-interacting) domain of the coactivator CBP (CREB Binding Protein). Methods: We assessed a library of previously untested alpha-mimetic compounds from ChemDiv designed to mimic interactions between α-helices and protein surfaces for predicted interaction with a shallow hydrophobic groove on the interaction surface of KIX. We interrogated the 24,737 compounds in the library using rigid-receptor ligand docking, followed by induced-fit ligand docking of the top compounds using two different software, MOE and Schrodinger's Bioluminate. The top 50 binding poses from each software's rankings were combined to yield 87 candidate alpha-mimetic molecule inhibitors. In addition to our screen of small molecules, we have designed peptide inhibitors that closely mimic the αB helix of pKID. We investigated two methods to enhance the stability and cell-penetrating ability of our peptides: hydrocarbon stapling and the utilization of a rigid scaffold from the scorpion toxin peptide BmBKTtx1. Based on the calculated binding scores and locations, the most promising chimeric peptide designs were further investigated for binding stability using 500 nanosecond molecular dynamics (MD) simulations modeling their interaction with KIX. Results: The binding poses of the final set of small molecules were visually inspected for occupation of key hotspots on the pKID:KIX interaction surface, including the location of the phosphorylation sight of KID. Of the 87 molecules and their binding poses, only 21 of them (24%) obstructed fewer than two of the three hotspots chosen for the analysis. Moreover, 80 of the molecules (92%) extended into the hydrophobic pocket, the most critical hotspot. The multi-residue construction of this pocket likely contributed to the high similarity in interaction fingerprint between these molecules and pKID; occupation of this pocket can serve as a good indicator of strong ligand binding to KIX in future assays. The most-promising peptide designs using the scorpion toxin scaffold were assessed with MD simulations and compared to a control peptide comprised primarily of the αB helix of pKID. The chimeric peptides showed increased binding stability over the control peptide with an average RMSD of 6.8Å vs. 12.5Å, illustrating the benefits of the stability afforded by the scorpion scaffold design. Several of the hydrocarbon stapled peptides were tested in vitro using fluorescence polarization (FP) assay developed by our lab (Chae et al. Bioorganic & Medicinal Chemistry Letters, 2019) and in AML HL-60 and KG-1 cells and were shown to have growth inhibitory effects by Cell-Titer Glo Assays. Discussion: These small molecules and scorpion toxin-derived peptides represent novel candidate inhibitors of the pKID:KIX interaction implicated in leukemia. Based on promising results from our in silico analyses of these inhibitors, we have obtained ten structurally diverse alpha-mimetic small molecules and three chimeric peptides and aim to test their efficacy in vitro using the FP assay. Furthermore, we will obtain high resolution cryo-EM structures of successful compounds and peptides bound to the KIX domain using a double-shell protein scaffold (Zhang et al. ACS Central Science, 2022), and use these structures for further refinement of the inhibitors. The development of new inhibitors for the pKID:KIX interaction promises to reveal insights into the structural relationship between the two domains and the basis for effective, targeted leukemia therapeutics.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Targeting Vimentin (VIM) in Acute Myelogenous Leukemia (AML)

Background: Vimentin (VIM) is a type III intermediate filament (IF) that anchors organelles in the cell, maintains cell integrity, and facilitates mitosis. A 2018 study demonstrated that the up-regulation of VIM is associated with poor clinical outcome in patients with Acute Myeloid Leukemia (AML). This study also reported that VIM regulates signaling networks that promote leukemic cell survival while conferring resistance to various stressors. A separate study revealed that VIM over-expression was associated both with markedly shorter overall survival (OS) and leukemia-free survival (LFS) in adult AML patients. VIM has been shown to be targetable by the steroidal lactone Withaferin-A which directly binds VIM, hyper-phosphorylates VIM's Serine 38 residue, and leads to an anti-proliferative effect characterized by perturbation of intermediary filaments. We investigated VIM expression and prognostic impact in pediatric AML, and whether inhibiting VIM affected chemosensitivity in vitro. Methods: Reverse Phase Protein Array (RPPA) was performed to quantify relative expression of 233 total and 63 post-translational modified proteins in 500 pediatric AML patients from the COG AAML1031 trial. AML cell lines with low VIM expression (OCI-2) and high VIM expression (OCI-3) as well as fresh, primary AML samples were exposed to the VIM inhibitor Withaferin-A alone, or in combination with Doxorubicin and Cytarabine chemotherapy. Other known VIM inhibitors including FiVe1, Simvastatin, and Ganoderic Acid A were tested for efficacy in our context, but only Withaferin-A produced a promising anti-leukemia effect. Results: In pediatric AML, VIM was over-expressed relative to normal CD34+ cells in all subtypes: bulk, CD3/CD19 depleted, CD34+, CD34+/38+, CD34+/38-, CD34-, CD34-/38+, and CD34-/38-. Pearson correlation analysis revealed significant negative correlations between VIM and proteins known to decrease during metastasis such as PDGFR-beta, TP15.pS15, HIF1-alpha and VHL. Elevated VIM correlated positively with HNRNPK, WTAP, ASH2L, and KDM1A, and H3K4Me2; alterations in histone methylation that are associated with metastatic potency. VIM expression was highest in FAB subtypes M4 and M5, and in patients with t(9;11) and MLL-rearrangements. VIM levels were lower in cytogenetically favorable and higher in unfavorable risk groups. Cumulative survival probability to standard chemotherapy progressively decreased as VIM expression increased (Fig. 1). We tested whether VIM inhibition with Withaferin-A at 50 nM and 100 nM, would affect proliferation or cell survival, finding a significantly stronger anti-proliferative effect (p<0.02) in the high-VIM OCI-3 cell line compared to the low-VIM OCI-2 cell line at 72 and 96 hours after treatment (Fig. 2). Combined with chemotherapy, Withaferin-A lead to greater cell death in the high-VIM OCI-3 cell line as opposed to low-VIM OCI-2 at 72 and 96 hours. For instance, in OCI-3, 100 nM Withaferin-A combined separately with either 1 uM Cytarabine or 50 nM Doxorubicin to decrease proliferation more than either chemotherapeutic agent alone (p=0.007 and p=0.0013). In primary AML samples, proliferation was slightly more inhibited at 72 hours post-treatment when Withaferin-A was paired with individual chemotherapeutics. Conclusions: Proteomic analysis demonstrated that VIM was elevated in numerous leukemia cell subtypes relative to normal CD34+ cells in children and adults and that prognosis worsened as VIM levels increased in pediatric AML. AML cell lines highly expressing VIM such as OCI-3 displayed sensitivity to both Withaferin-A monotherapy and Withaferin-A in combination with Cytarabine and Doxorubicin. Finally, Withaferin-A significantly reduced viability and proliferation after 72 hours of treatment in primary AML samples at select doses in combination with chemotherapeutics. These results suggest that targeting VIM could improve the efficacy of chemotherapy in high VIM expressing subpopulations and potentially improve overall survival. Withaferin-A's efficacy both as a mono-therapeutic and in combination with standard chemotherapeutics used to treat AML patients offers an exciting new area of research that requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal