Therapeutic Target In Acute Myeloid Leukemia Research Articles

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

Prognostic and therapeutic implications of iron-related cell death pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is diverse in terms of its molecular abnormalities and clinical outcomes. Iron homeostasis and cell death pathways play crucial roles in cancer pathogenesis, including AML. The objective of this study was to examine the clinical significance of genes involved in iron-related cell death and apoptotic pathways in AML, with the intention of providing insights that could have prognostic implications and facilitate the development of targeted therapeutic interventions. Gene expression profiles, clinical information, and molecular alterations were integrated from multiple datasets, including TCGA-LAML and GSE71014. Our analysis identified specific molecular subtypes of acute myeloid leukemia (AML) displaying varying outcomes, patterns of immune cell infiltration, and profiles of drug sensitivity for targeted therapies based on the expression of genes involved in iron-related apoptotic and cell death pathways. We further developed a risk model based on four genes, which demonstrated promising prognostic value in both the training and validation cohorts, indicating the potential of this model for clinical decision-making and risk stratification in AML. Subsequently, Western blot analysis showed that the expression levels of C-Myc and CyclinD1 were significantly reduced after CD4 expression levels were knocked down. The findings underscore the potential of iron-related cell death pathways as prognostic biomarkers and therapeutic targets in AML, paving the way for further research aimed at understanding the molecular mechanisms underlying the correlation between iron balance, apoptosis regulation, and immune modulation in the bone marrow microenvironment.

The prognostic value of 19S ATPase proteasome subunits in acute myeloid leukemia and other forms of cancer.

The ubiquitin-proteasome system (UPS) is an intracellular organelle responsible for targeted protein degradation, which represents a standard therapeutic target for many different human malignancies. Bortezomib, a reversible inhibitor of chymotrypsin-like proteasome activity, was first approved by the FDA in 2003 to treat multiple myeloma and is now used to treat a number of different cancers, including relapsed mantle cell lymphoma, diffuse large B-cell lymphoma, colorectal cancer, and thyroid carcinoma. Despite the success, bortezomib and other proteasome inhibitors are subject to severe side effects, and ultimately, drug resistance. We recently reported an oncogenic role for non-ATPase members of the 19S proteasome in chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and several different solid tumors. In the present study, we hypothesized that ATPase members of the 19S proteasome would also serve as biomarkers and putative therapeutic targets in AML and multiple other cancers. We used data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) available at UALCAN and/or GEPIA2 to assess the expression and prognostic value of proteasome 26S subunit, ATPases 1-6 (PSMC1-6) of the 19S proteasome in cancer. UALCAN was also used to associate PSMC1-6 mRNA expression with distinct clinicopathological features. Finally, cBioPortal was employed to assess genomic alterations of PSMC genes across different cancer types. The mRNA and protein expression of PSMC1-6 of the 19S proteasome were elevated in several cancers compared with normal controls, which often correlated with worse overall survival. In contrast, AML patients demonstrated reduced expression of these proteasome subunits compared with normal mononuclear cells. However, AML patients with high expression of PSMC2-5 had worse outcomes. Altogether, our data suggest that components of the 19S proteasome could serve as prognostic biomarkers and novel therapeutic targets in AML and several other human malignancies.

Apicidin confers promising therapeutic effect on acute myeloid leukemia cells via increasing QPCT expression

ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by abnormal cell proliferation, apoptosis repression and myeloid differentiation blockade of hematopoietic stem/progenitor cells. Developing and identifying novel therapeutic agents to reverse the pathological processes of AML are of great significance. Here in this study, we found that a fungus-derived histone deacetylase inhibitor, Apicidin, presents promising therapeutic effect on AML by inhibiting cell proliferation, facilitating apoptosis and inducing myeloid differentiation of AML cells. Mechanistic investigation revealed that QPCT is identified as a potential downstream target of Apicidin, which exhibits significantly decreased expression in AML samples compared with the normal controls and is remarkably up-regulated in AML cells upon Apicidin management. Functional study and rescue assay demonstrated that QPCT depletion further promotes cell proliferation, inhibits apoptosis and impairs myeloid differentiation of AML cells, alleviating the anti-leukemic effect of Apicidin on AML. Our findings not only provide novel therapeutic target for AML, but also lay theoretical and experimental foundation for the clinical application of Apicidin in AML patients.

Classification of Acute Myeloid Leukemia by Cell-Free DNA 5-Hydroxymethylcytosine.

Epigenetic abnormality is a hallmark of acute myeloid leukemia (AML), and aberrant 5-hydroxymethylcytosine (5hmC) levels are commonly observed in AML patients. As epigenetic subgroups of AML correlate with different clinical outcomes, we investigated whether plasma cell-free DNA (cfDNA) 5hmC could categorize AML patients into subtypes. We profiled the genome-wide landscape of 5hmC in plasma cfDNA from 54 AML patients. Using an unbiased clustering approach, we found that 5hmC levels in genomic regions with a histone mark H3K4me3 classified AML samples into three distinct clusters that were significantly associated with leukemia burden and survival. Cluster 3 showed the highest leukemia burden, the shortest overall survival of patients, and the lowest 5hmC levels in the TET2 promoter. 5hmC levels in the TET2 promoter could represent TET2 activity resulting from mutations in DNA demethylation genes and other factors. The novel genes and key signaling pathways associated with aberrant 5hmC patterns could add to our understanding of DNA hydroxymethylation and highlight the potential therapeutic targets in AML. Our results identify a novel 5hmC-based AML classification system and further underscore cfDNA 5hmC as a highly sensitive marker for AML.

Prolactin receptor signaling induces acquisition of chemoresistance and reduces clonogenicity in acute myeloid leukemia

BackgroundDevelopment of precision medicine requires the identification of easily detectable and druggable biomarkers. Despite recent targeted drug approvals, prognosis of acute myeloid leukemia (AML) patients needs to be greatly improved, as relapse and refractory disease are still difficult to manage. Thus, new therapeutic approaches are needed. Based on in silico-generated preliminary data and the literature, the role of the prolactin (PRL)-mediated signaling was interrogated in AML.MethodsProtein expression and cell viability were determined by flow cytometry. Repopulation capacity was studied in murine xenotransplantation assays. Gene expression was measured by qPCR and luciferase-reporters. SA-β-Gal staining was used as a senescence marker.ResultsThe prolactin receptor (PRLR) was upregulated in AML cells, as compared to their healthy counterpart. The genetic and molecular inhibition of this receptor reduced the colony-forming potential. Disruption of the PRLR signaling, either using a mutant PRL or a dominant-negative isoform of PRLR, reduced the leukemia burden in vivo, in xenotransplantation assays. The expression levels of PRLR directly correlated with resistance to cytarabine. Indeed, acquired cytarabine resistance was accompanied with the induction of PRLR surface expression. The signaling associated to PRLR in AML was mainly mediated by Stat5, in contrast to the residual function of Stat3. In concordance, Stat5 mRNA was significantly overexpressed at mRNA levels in relapse AML samples. A senescence-like phenotype, measured by SA-β-gal staining, was induced upon enforced expression of PRLR in AML cells, partially dependent on ATR. Similar to the previously described chemoresistance-induced senescence in AML, no cell cycle arrest was observed. Additionally, the therapeutic potential of PRLR in AML was genetically validated.ConclusionsThese results support the role of PRLR as a therapeutic target for AML and the further development of drug discovery programs searching for specific PRLR inhibitors.

Circular RNAs: pivotal role in the leukemogenesis and novel indicators for the diagnosis and prognosis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy and affected patients have poor overall survival (OS) rates. Circular RNAs (circRNAs) are a novel class of non-coding RNAs (ncRNAs) with a unique loop structure. In recent years, with the development of high-throughput RNA sequencing, many circRNAs have been identified exhibiting either up-regulation or down-regulation in AML patients compared with healthy controls. Recent studies have reported that circRNAs regulate leukemia cell proliferation, stemness, and apoptosis, both positively and negatively. Additionally, circRNAs could be promising biomarkers and therapeutic targets in AML. In this study, we present a comprehensive review of the regulatory roles and potentials of a number of dysregulated circRNAs in AML.

Abstract 1732: Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment

Abstract CD36 has been identified as a potential therapeutic target both in the leukemic cells and in the tumor immune microenvironment. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. To establish CD36 as a viable therapeutic target in AML, we recently investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. We compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (Cd36-KO) with C57BL/6J control (WT) mice (N=6 per group). Cd36-KO mice have normal blood counts except for lower red blood cells count (5.85 vs 6.93 (x 106/μL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) than WT mice, yet these values remained within the normal range. Cd36-KO mice exhibited similar spleen T cell phenotypes compared with the WT mice and expanded similarly in vitro in the presence IL2 and PHA. The in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSPC expansion media containing SCF and TPO cytokines was also similar between Cd36-KO mice and WT mice. However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of Cd36-KO mice resulted in 40% fewer colonies than WT mouse HSPC cells (P = 0.0003). However, when HSPC obtained from Cd36-KO and WT mice were transplanted in CD45.1 mice, their percentage of engraftment were not significantly different in the bone marrow (BM: KO vs WT: 64.92% vs 63.45%, P > 0.9999), spleen (40.88% vs 40.05%, P = 0.8413), and blood (83.47% vs 82.88%, P > 0.9999). To assess whether CD36 in the leukemia microenvironment has an impact on leukemia growth and progression, we injected 5 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells via tail vein in either Cd36-KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36-KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Cd36-KO and WT mice developed similar leukemia burden when less aggressive models are used with the engraftment of 1 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells. Altogether, our preliminary data suggest that loss of Cd36 may influence the hematopoietic stem cell and erythropoiesis yet have limited detrimental overall impact on normal hematopoiesis and leukemia microenvironment. Therefore, targeting CD36 in leukemic cells presents limited toxicity on normal hematopoiesis and therapeutic approaches to target this receptor present safe strategies to treat AML. Citation Format: Yiting Meng, Mateusz Pospiech, Atham Ali, Ritu Chandwani, Sandra Onyemaechi, George Yaghmour, Houda Alachkar. Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment [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 1732.

Loss of IRF8 inhibits the growth of acute myeloid leukemia cells.

The transcription factor interferon regulatory factor 8 (IRF8), as a member of the IRF family, is essential for myeloid cell differentiation. However, the precise role of IRF8 in the pathogenesis of acute myeloid leukemia (AML) remains unknown. By using multivariate analysis, we discovered that high IRF8 expression was an independent poor predictor of overall survival (OS) in AML patients from our clinical follow-up study. The proliferation of three AML cell lines was significantly inhibited by shRNA-mediated knockdown of IRF8, owing to cell cycle S-phase arrest. Furthermore, we demonstrated that knocking down IRF8 could suppress the expression of CyclinA and CyclinB1, resulting in a shift in cell cycle distribution. Loss of IRF8 in AML cells decreased the expression of STAT3 and phosphor-STAT3 (pSTAT3), which are key factors in JAK/STAT signal pathway and are important for AML progression. Using a xenograft mouse model, we discovered the antiproliferative effect of losing IRF8 in vivo. In conclusion, this study found that IRF8 may play a prognostic factor and therapeutic target in AML.

In vivo screening unveils pervasive RNA-binding protein dependencies in leukemic stem cells and identifies ELAVL1 as a therapeutic target.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSCs) whose determinants are challenging to discern from hematopoietic stem cells (HSCs) or uncover by approaches focused on general cell properties. We have identified a set of RNA binding proteins (RBPs) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution and selectively depleted primitive malignant vs. healthy cells. Integrative multiomics revealed differentiation, splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34 being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.

High GLI-1 Expression is a Reliable Indicator of Bad Prognosis in Newly Diagnosed Acute Leukemia Patients

Purpose: To explore the expression and prognostic significance of Hedgehog signaling transcription factor GLI-1 in newly diagnosed acute myeloid leukemia (AML) patients. Methods: Clinical specimens were obtained from 46 recently diagnosed AML patients. Real-time qPCR was used to measure the GLI-1 mRNA expression in bone marrow mononuclear cells.Also, the relationship between GLI-1 mRNA levels and clinical variables and prognostic variables was assessed. Results: GLI-1 was overexpressed in the bone marrow samples of our patients. GLI-1mRNA expression did not differ significantly across different age groups, between both sexes, or between different FAB subtypes (P = 0.882, P = 0.246, and P = 0.890, respectively). GLI-1 expression varied significantly in different risk categories, with the greatest levels observed in 11 patients with poor risk (24.6 versus 22.7) compared to intermediate risk (5.2 versus 3.9; P = 0.006) and favorable risk (4.2 versus 3; P = 0.001). Comparing patients with the wild FLT3 allele to those with the mutant one, GLI-1 gene levels were considerably greater in those with the mutant allele of FLT3.Following induction chemotherapy, the levels of GLI-1 mRNA were significantly higher in 22 patients who did not experience complete remission (CR) diagnosed with de novo non-acute promyelocytic leukemia (APL) compared to 17 patients who did (P = 0.017). Significantly greater levels of expression were observed in each category of the patients with favorable risk; wild FLT3 allele (P = 0.033) and CR failure P = 0.005). Conclusion: GLI-1 overexpression is a risk factor for poor prognosis and could be a novel therapeutic target for AML.

ZNF460-regulated COMMD7 Promotes Acute Myeloid Leukemia Proliferation Via the NF-κB Signaling Pathway.

Acute myeloid leukemia (AML) is a malignancy of the hematological system, for which there remains an urgent need for new therapeutic and diagnostic targets. COMM domain containing 7 (COMMD7) is a recently-identified oncogene linked to poor prognosis in AML. COMMD7 regulates multiple signaling pathways, including nuclear factor-kappa B (NF-κB) signaling. Here, we report that COMMD7 is highly expressed in the AML cell lines KG1a and U937 and that its inhibition by shRNA reduced proliferation, promoted apoptosis and facilitated cell cycle arrest in the G2/M phase in relation to depression of the NF-κB pathway. Furthermore, zinc finger protein 460 (ZNF460) is overexpressed in AML and regulates COMMD7. We found that knockdown of ZNF460 downregulated the expression of COMMD7 while the NF-κB pathway was also inhibited. In addition, we noticed that knockdown of ZNF460 reduced proliferation and increased apoptosis rate of AML cells and that the cell cycle was blocked in the G2/M phase. In brief, our results revealed a critical effect of the ZNF460-COMMD7-NF-κB axis for the proliferation of AML cells. Therefore, COMMD7 may be a possible therapeutic target for AML.

Inhibition of Stearoyl-CoA Desaturase Has Anti-Leukemic Properties in Acute Myeloid Leukemia

Metabolic rewiring, a hallmark of acute myeloid leukemia (AML), contributes to the development of resistance to intensive chemotherapy and has been shown to play an essential role in leukemic cell maintenance. Several metabolic alterations have been described in AML, such as dependence on amino acids to fuel oxidative phosphorylation or ability of leukemic cells to switch energy dependence to fatty acid oxidation in response to treatment. However, little is known about the functional significance of the fatty acid synthesis (FAS) pathway in AML. Here we have identified stearoyl-CoA desaturase (SCD), a key enzyme responsible for the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), as a novel therapeutic target in AML. High SCD expression predicts worse survival across several patient datasets, even after correction for gender, age and AML European LeukemiaNet risk category. Moreover, genetic and chemical inhibition of SCD reduces growth and induces cell death in AML cells. Since targeting SCD with currently available inhibitors has been challenging because of their toxicity profile in vivo, we have accessed a novel pharmacological SCD inhibitor with a more favorable toxicity profile, SSI-4 (Modulation Therapeutics Inc., Morgantown, USA). SSI-4 reduces viability in several human and mouse AML models in vitro and in about half of the 36 primary AML samples tested ex vivo. Moreover, SSI-4 displays anti-leukemic effects in AML murine models and patient-derived xenografts (PDX). Since we could not correlate susceptibility to SCD inhibition with any genetic characteristic of AML cells, we aimed to determine the molecular basis of cell sensitivity using phosphoproteomic, proteomic and transcriptomic approaches coupled with metabolic analyses of sensitive and resistant cell lines and primary AML cells. Sensitivity to SCD inhibition directly correlates with rate of de novo FAS, as measured by fatty acid labeling with 13C-glucose. Moreover, modulation of FAS by the AMPK signaling activity dictates sensitivity to SCD inhibition and AMPK activation rescues the anti-leukemic effects of SSI-4. The anti-leukemic effects of SCD inhibition are secondary to the induction of SFA mediated lipotoxicity, as demonstrated by the increased sensitivity of previously resistant cells to SSI-4, when grown in the presence of the SFA palmitate. Conversely, growing cells in the presence of the MUFA oleate, as well as inhibition of FAS by targeting enzymes upstream of SCD, results in resistance to SSI-4, thus confirming that the increased SFA/MUFA ratio drives leukemic cell death. SFA mediated lipotoxicity leads to both increased lipid peroxidation and activation of the unfolded protein response (UPR), namely its PERK branch, which in turn leads to apoptotic cell death. Indeed, inhibition of PERK, but not of other branches of UPR, rescues SCD-mediated cytotoxicity. Despite causing disease debulking in vivo, SSI-4 does not prolong survival in murine AML models as a single agent and the toxic effects of SSI-4 are decreased when AML cells are grown in co-culture with stroma, suggesting a protective role of tumor microenvironment. However, we consistently detect increased lipid peroxidation and DNA damage both in vitro and in vivo following SCD inhibition. We therefore combined SSI-4 with doxorubicin, a known inducer of lipid peroxidation and DNA damage, which is routinely used in AML induction therapy. Indeed, both genetic depletion and pharmacological inhibition of SCD renders AML cells more sensitive to doxorubicin, and addition of SSI-4 to a standard AML chemotherapy protocol consisting of doxorubicin and cytarabine significantly extends survival in AML mouse models. In summary, we identify and validate SCD as a therapeutic target in AML. The novel SCD inhibitor SSI-4 displays anti-leukemic properties both in vitro and in vivo across different AML models and particularly when combined with standard chemotherapy while showing minimal toxicity in vivo. We show that sensitivity to SCD inhibition is driven by metabolic rather than genetic AML features, thus making this therapeutic approach potentially applicable across several AML subtypes, including high risk ones. Finally, the mechanism of toxicity following SCD inhibition suggests that this can be further enhanced by modulating the levels of SFA/MUFA through dietary interventions or by combining it with DNA damaging agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD123 Directed IgM Antibody-based T-cell Engager, IGM-2537, Demonstrates Potent in vitro and in vivo Activity with Minimal Cytokine Release

Introduction: Despite recent progress, the outlook for patients with acute myeloid leukemia (AML) remains poor. Novel therapies are needed for effective treatment of AML, especially in the relapsed/refractory setting. Evidence suggests that incomplete depletion of leukemia stem cells (LSCs) causes relapse. CD123, the IL-3 receptor alpha chain, is highly expressed on both leukemic blasts and LSCs and thus is a suitable therapeutic target in AML. CD123-based immunotherapies including bispecific antibodies or CAR T cells have shown promise, but cytokine release syndrome (CRS) is a major safety concern with dose limiting toxicity (DLT). We are developing IGM-2537, a novel IgM antibody-based CD123xCD3 bispecific T cell engager (TCE), designed with potent anti-tumor activity and lower cytokine release. IGM-2537 has 10 binding sites for CD123, and single binding site for CD3 via single chain Fv domain (scFv) fused to a joining (J) chain, thus providing high affinity, high avidity binding to CD123-positive malignant cells while co-engagement of CD3 T cells leading to T cell induced cytotoxicity with reduced cytokine release. Here we report functional characterization of IGM-2537 in a broad range of in vitro, ex vivo and in vivo anti-tumor efficacy studies. The improved tolerability, good pharmacodynamic (PD) response and lower cytokine release profile of a benchmark IgM TCE comparator in cynomolgus monkeys will be discussed. Materials and Methods: IGM-2537 was constructed using the pentameric human IgM framework. Recombinant IgM heavy chains and light chains with high affinity variable domains derived from an anti-CD123 IgG were co-expressed with a J-chain fused to anti-CD3 scFv. Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity, and epitope mapping, respectively. Bispecific enzyme-linked immunosorbent assay (ELISA), T-cell NFAT reporter assay and T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 to co-engage both CD123 and CD3 antigens, to induce T cell activation and target cytotoxicity, respectively. Ex vivo AML colony formation assay was applied to assess its activity to eliminate AML blasts. Human MV4-11 xenograft tumor model in humanized NSGdKO mice were utilized to determine anti-tumor efficacy of IGM-2537. Lastly, to confirm that CD123 directed IgM-based TCE molecule can potentially provide better safety profile, a benchmark IgM TCE comparator was assessed in cynomolgus monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound to human CD123 with high affinity, avidity, and specificity. Furthermore, IGM-2537 bound a unique epitope of human CD123 in domain 2 which does not overlap with IL-3. IGM-2537 co-engaged with both CD123 and CD3 antigens, leading to T cell-redirected killing of AML cell lines with concomitant T cell activation. Moreover, IGM-2537 eliminated AML blast cells at physiologically relevant effector/target (E/T) ratios in an ex vivo assay. Most importantly, IGM-2537 demonstrated comparable maximal killing activity to a benchmark IgG TCE molecule but shows significant reduction of cytokine release exemplified by IFN-γ, TNF-α and IL-6, which provide initial evidence of better preclinical safety profile for a CD123 directed IgM-based T-cell engager. As expected, the efficient in vitro TDCC activity of IGM-2537 translated into in vivo anti-tumor efficacy with tumor growth inhibition observed at dose ranges from 1 to 10 mg/kg. To further support that the IgM TCE format can provide a better safety window, a benchmark IgM TCE comparator was evaluated for tolerability (including cytokine production), PK and PD responses in cynomolgus monkeys. All animals tolerated the benchmark comparator well at dose levels up to 10 mg/kg (the maximal dose level evaluated); however, the tolerability observed with IgM TCE was 100x greater compared to IgG TCE. Complete depletion of CD123-positive basophils and substantial reduction of plasmacytoid dendritic cells (pDC) were seen in blood and bone marrow with minimal to no cytokine induction. In summary, our IGM-2537 demonstrates potent in vitro and in vivo activity with minimal cytokine induction. IgM as the framework of a TCE molecule can potentially provide better safety profile and an improved therapeutic window.

Anagrelide Represents a Potential Therapeutic Therapy for Acute Myeloid Leukemia Cells Based on Phosphodiesterase 3A Expression

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. PDE3A is a well-characterized cyclic nucleotide phosphodiesterase (PDE) that hydrolyzes cAMP, cGMP, and cUMP. Recent studies have demonstrated that PDE3A was upregulated in several solid tumors of patients including breast cancer, ovarian carcinoma, melanoma, which correlated with poor prognosis. PDE3A activates inflammatory pathways that mediate cancer cell stemness by suppressing the cAMP/PKA pathway. Targeting PDE3A with chemicals suppressed cancer cells by inducing cell apoptosis and reducing metastasis. PDE3A inhibitor Anagrelide (ANA) is an antithrombotic and platelet reducing agent used to treat the thrombocythemia associated with myeloproliferative diseases. This study aims to explore the relationship between PDE3A expression and AML prognosis, and to investigate the feasibility and mechanism of PDE3A as a therapeutic target for AML. To explore the relationship between PDE3A expression and the survival time of patients, a total of 141 pediatric AML patients were recruited in this retrospective study from October 2013 to September 2021 in the Children's Hospital of Soochow University. The follow-up period was until March 2022. The transcriptomic expression level of PDE3A in newly diagnosed bone marrow samples was obtained according to the results of RNA sequencing. Patients were divided into two groups based on median PDE3A expression: PDE3Ahigh group (n= 70) and PDE3Alow group(n=71). Results showed AML patients in PDE3Ahigh group had significantly lower 5-year overall survival (OS) (71.4% vs 92.9%, P = 0.0010), event free survival (52.1% vs 84.6%, P = 0.0150) and relapse free survival (73.2% vs 91.4%, P = 0.0325) compared to patients in PDE3Alow group. Next, to evaluate PDE3A expression level in AML cell lines, we used RT-PCR and Western Blot to compare the difference of PDE3A expression in HEL, Kasumi-1, MV4-11, NB4, K562, HL60 and MEG-01. Among these cell lines, HEL has significantly higher PDE3A expression compared to others at both transcriptional and protein levels. We found that ANA specifically inhibited HEL growth in a concentration dependent manner after 72 hours of ANA treatment. ANA reached half of the inhibitory concentration on HEL at 0.9μM. Interestingly, ANA had no inhibitory effects on other tested AML cell lines which expressed low level of PDE3A. Thus, we choose HEL as PDE3Ahigh AML cell model and treatment with 0.9μM ANA for 72 hours for further experiments. By flow cytometry, we found ANA interrupted cell cycle by G1 arrest in HEL, but did not induce cell apoptosis. In consistent with these results, the expression of cell cycle-related proteins including CDK2, CDK4, CDK6, CDK9 and C-MYC were significantly down-regulated in a time-dependent manner after ANA treatment in HEL compared to control group. To explore the effect of ANA on PDE3A high expression AML in vivo, we established the preclinical model of AML by injecting 2 million luciferase overexpressed HEL cells into NSG mice via tail vein. Fourteen days later, the mice were treated with vehicle or 5mg/kg ANA treatment by gavage daily for 15 days as the therapeutic model. More interestingly, in vivo imaging assay indicated that ANA treatment reduced leukemia burden compared with the vehicle group. The anti-leukemia effects of ANA significantly prolonged survival time compared to vehicle (median survival time, 32.5 vs 24.5 days, P = 0.0091). In conclusion, our clinical data suggested that high expression of PDE3A in newly diagnosed pediatric AML patients was associated with poor clinical survival. Besides, our in vitro and in vivo data indicated that ANA targeting PDE3A suppressed progression of AML by interrupting cell cycle, providing a promising approach to eradicate AML with high level of PDE3A expression.

Activating a Non-Genetic MYC-Regulating Pathway Sensitizes Acute Myeloid Leukemia to Bromodomain Inhibition

MYC (c-Myc) plays a central role in the oncogenic progression and is commonly overexpressed or amplified in more than 50% of human cancers, including acute myeloid leukemia (AML), which is an aggressive disease with over 70% of patients who cannot survive more than five years. Elevated MYC expression is associated with therapeutic resistance and predicts a poorer outcome in AML patients. While numerous lines of evidence have validated MYC as a therapeutic target of AML and many other cancer types, MYC protein itself has been a challenge for therapeutic targeting. Recently, MYC gene transcription has been linked to the bromodomain and extra-terminal domain (BET) family proteins. BET inhibitors (BETi) inhibit MYC expression and have shown promising antitumor activities in preclinical models and are being evaluated in clinical trials. Despite the therapeutic promise, BETi-associated drug resistance and dose-limiting side effects hinder the clinical applications. Therefore, there is a pressing need to understand the mechanisms controlling drug response and resistance to BET inhibitors. To understand the mechanisms that regulate BETi sensitivity, we leveraged a functional CRISPR screen focusing on about 1,700 human transcription factors. We discovered the aryl hydrocarbon receptor (AHR) as a novel regulator of BETi sensitivity in AML. Our results have further identified that a novel AHR-ELMSAN1 axis is critically involved in regulating MYC and BETi sensitivity. Specifically, we show that constitutive or ligand activation of AHR sensitizes leukemia cells to BETi. AHR signaling represses MYC and synergizes with BETi for maximum inhibition of MYC transcription. Ectopic expression of MYC could rescue the anti-leukemia effects induced by combined AHR activation and BET blockade. Mechanistically, our unbiased bioinformatic analysis unexpectedly identified that AHR functions through activating a non-canonical target, ELMSAN1, a less-studied epigenetic factor known as a member of the MiDAC histone deacetylase complex. Depletion of ELMSAN1 leads to upregulation of MYC and blunts AHR signaling-mediated MYC repression and BETi sensitization. Importantly, we demonstrate that AHR agonist and BETi synergistically inhibit MYC and leukemia cell growth in CD34+ primary AML patient samples in vitro and patient-derived xenografts in vivo. In summary, our data suggest that AHR-ELMSAN1 is a novel pathway for MYC repression, activation of which could sensitize AML to BET-targeting agents. As neither AHR nor ELMSAN1 is found mutated in AML, our research may open a new door to new MYC targeting paradigms by activating novel non-genetic MYC repressors.

Selected Articles from This Issue

Mutant NRASG12V drives acute myeloid leukemia (AML) cell proliferation and self-renewal in a mutually exclusive manner. Previous studies demonstrated that AML cell proliferation and self-renewal are associated with differential NRASG12V transcript levels, but whether different NRASG12V protein levels dictate proliferative versus self-renewal phenotypes is not known. To address that knowledge gap, Kurata and colleagues engineered AML cell lines in which tetracycline-inducible NRASG12V expression levels are tunable using various doxycycline doses. The authors found that moderate NRASG12V protein expression promotes AML cell proliferation, whereas higher NRASG12V protein expression facilitates AML cell senescence and self-renewal. Using mass cytometry, the authors showed that abundances of several downstream NRASG12V effectors, including known stemness mediators such as β-catenin, increase in a dose-dependent fashion, whereas others, such as phosphorylated STAT1, are not dose-dependent. Overall, this study demonstrates that differential NRASG12V protein levels disparately drive AML cell proliferation and self-renewal and illuminates the downstream signaling dynamics underlying each phenotype.Tumor cell heterogeneity is a major impedance in eliminating cancer, as it promotes therapy-resistant clone selection in response to treatment. Identifying biomarkers indicative of tumor heterogeneity may help quantify it and uncover therapeutic vulnerabilities, but such biomarkers remain elusive. In their study, So and colleagues discovered that DNA methyltransferase 3B (DNMT3B) protein expression displays marked heterogeneity in primary tumors from triple-negative breast cancer (TNBC) patients and TNBC mouse models. By selecting DNMT3B-high and -low clones from 4T1 cells, labeling them with different fluorescent dyes, and quantifying them in circulation and lung metastases after orthotopic injection, the authors found that DNMT3B-high cells are more metastatic than DNMT3B-low cells. Mechanistically, prostaglandin E2 (PGE2) induces DNMT3B expression, and DNMT3B regulates expression of epithelial–mesenchymal transition effectors including vimentin, Twist2, and Snai1. Perioperative treatment with celecoxib, which lowers PGE2 expression, alongside DNMT inhibitor 5-azacytidine and other chemotherapeutic drugs decreases tumor recurrence and metastasis after primary tumor removal. Taken together, this study presents DNMT3B as a therapeutically targetable marker and effector of tumor heterogeneity.Therapeutic challenges and poor outcomes in pancreatic cancer make further elucidating its underlying molecular mechanisms and therapeutic vulnerabilities critical. While long non-coding RNA (lncRNA) α-2-macroglobulin-antisense 1 (A2M-AS1) expression positively correlates with overall survival in pancreatic cancer as well as ferroptosis-related genes, how A2M-AS1 might regulate ferroptosis and whether it does so in pancreatic cancer has not been solved. In their study, Qiu and colleagues found that pharmacologically inducing ferroptosis in pancreatic cell lines augments A2M-AS1 expression, and that modulating A2M-AS1 abundance using shRNA and overexpression inhibits and enhances ferroptosis, respectively. Accordingly, A2M-AS1 overexpression limits pancreatic cancer cell growth both in vitro and in vivo. An RNA pull-down and mass spectrometry revealed that A2M-AS1 interacts with PCBP3. Additional experiments demonstrated that the interaction between A2M-AS1 and PCBP3 abrogates mTOR and AKT activation, while increasing p38 activation and ferroptosis. In sum, this study unveils a novel molecular mechanism by which ferroptosis evasion occurs in pancreatic cancer.Acute myeloid leukemia (AML) cells are dependent on oxidative metabolism. However, electron transport chain complex II (CII) targeting agents and their potential effects on AML cell metabolism and viability have not been assessed. To evaluate CII therapeutic targeting in AML, Roma and colleagues abrogated expression of CII chaperone protein succinate dehydrogenase assembly factor 1 (SDHAF1) using shRNA in AML cell lines. Using an in silico screen and HPLC analysis, the authors also identified shikonin as a CII small molecule inhibitor. Both SDHAF1 ablation and shikonin promote AML cell death in vitro and in vivo, and stable isotope tracing demonstrated concurrent tricarboxylic acid (TCA) cycle truncation. The authors also showed that SDHAF1 ablation and shikonin increase glutamine flux into the TCA cycle via deamination to α-ketoglutarate as well as reductive flux to aspartate. Accordingly, supplementing α-ketoglutarate undermines shikonin effectiveness, and pharmacologically inhibiting glutaminase augments it. Altogether, this study reveals a novel therapeutic target and accompanying therapeutic agent in AML and identifies anaplerotic glutamine metabolism as a compensatory mechanism and therapeutic vulnerability in CII inhibition.

A novel 10-gene ferroptosis-related prognostic signature in acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies and exhibits a high rate of relapse and unfavorable outcomes. Ferroptosis, a relatively recently described type of cell death, has been reported to be involved in cancer development. However, the prognostic value of ferroptosis-related genes (FRGs) in AML remains unclear. In this study, we found 54 differentially expressed ferroptosis-related genes (DEFRGs) between AML and normal marrow tissues. 18 of 54 DEFRGs were correlated with overall survival (OS) (P<0.05). Using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis, we selected 10 DEFRGs that were associated with OS to build a prognostic signature. Data from AML patients from the International Cancer Genome Consortium (ICGC) cohort as well as the First Affiliated Hospital of Wenzhou Medical University (FAHWMU) cohort were used for validation. Notably, the prognostic survival analyses of this signature passed with a significant margin, and the riskscore was identified as an independent prognostic marker using Cox regression analyses. Then we used a machine learning method (SHAP) to judge the importance of each feature in this 10-gene signature. Riskscore was shown to have the highest correlation with this 10-gene signature compared with each gene in this signature. Further studies showed that AML was significantly associated with immune cell infiltration. In addition, drug-sensitive analysis showed that 8 drugs may be beneficial for treatment of AML. Finally, the expressions of 10 genes in this signature were verified by real-time quantitative polymerase chain reaction. In conclusion, our study establishes a novel 10-gene prognostic risk signature based on ferroptosis-related genes for AML patients and FRGs may be novel therapeutic targets for AML.

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway.

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G2/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.

The presence of Auer rods in neutrophils and monocytes besides blasts in a case of acute myeloid leukemia with CSF3R mutation.

An 81-year-old male with a history of diabetes, hypertension, hypercholesterolemia, previous DVT not on anticoagulation, and arthritis presented to the emergency department with a chief complaint of generalized weakness. Complete blood counts revealed white blood cells (WBC) 165.3 × 109/L, hemoglobin (Hb) 4.4 g/dl, and platelet (PLT) < 10 × 109/L, with 62% blasts. Review of peripheral blood showed blasts of variable sizes with a moderate amount of basophilic cytoplasm (Figure 1A–D; peripheral blood; Leishman stain; 100× objective), and pale-salmon-pink-colored cytoplasmic granules in a subset (Figure 1A, see arrows). Auer rods are easily identified in blasts (Figure 1A,B, see asterisk), occasionally seen in monocytes (Figure 1C, see asterisk) and neutrophils (Figure 1d, see asterisk). Bone marrow was examined to establish diagnosis, showing marked hypercellularity with 72% blasts on aspirate smears with morphologic features similar to those in peripheral blood (Figure 1E [bone marrow biopsy; hematoxylin and eosin, 40× objective] and F [bone marrow aspirate; Leishman stain; 50× objective]). Granulocytic dysplasia with pelgeroid morphology and abnormal nuclear chromatin clumping was seen in both peripheral blood and bone marrow (> 10% and < 50%). Concurrent flow cytometry showed increased myeloblasts with expression of CD34, CD13, CD 117, CD33, HLA-DR, CD19 (dim; minor subset), CD11b (dim; subset), CD14 (dim; minor subset), CD15 (dim; minor subset), myeloperoxidase (dim; subset), and terminal deoxynucleotidyl transferase (dim; minor subset). Conventional cytogenetics showed normal male karyotype. Concurrent FISH studies using acute myeloid leukemia (AML) and myelodysplastic syndrome panels of probes are negative for all loci studied, including 8q22 and 21q22 probes for RUNX1-RUNX1T1 rearrangement. Next generation sequencing showed mutation on the CSF3R gene (p.T640N, variant allele frequency 48.4%). An integrated diagnosis of AML with maturation was established (French–American–British Classification Subtype M2). Auer rods are a morphologic feature in leukemic blasts defining myeloid differentiation. The presence of Auer rods in neutrophils and monocytes are rarely reported, most commonly in association with acute promyelocytic leukemia (APL) with PML-RARA and AML with RUNX1-RUNX1T1, occasionally seen in other subtypes of AML and myelodysplastic syndrome (MDS) in the literature as well [1]. We presented a case of AML with Auer rods in non-blast cells as well as morphologic and immunophenotypic features resembling AML with RUNX1-RUNX1T1: blasts with basophilic cytoplasm, salmon-pink (pale) cytoplasmic granules and perinuclear Hof besides Auer rods as well as granulocytic dysplasia in the background. However, no recurrent cytogenetic and molecular genetic abnormalities as defined by World Health Organization (WHO) classification are detected, while a CSF3R mutation seen. Although mutations in the colony stimulating factor 3 receptor (CSF3R) occurs rarely in AML, comprising only about 1% of adults with AML, it is suggested that CSF3R can be a potential therapeutic target in AML [2]. In summary, we presented for the first time a case of AML with a few rare morphologic features including the presence of Auer rods in non-blast cells coinciding with a potential therapeutic target of CSF3R mutation. Whether the association between the morphologic features and the CSF3R mutation can be established warrants additional studies. In addition, a potential diagnostic pitfall to be taken into consideration in this case is that no reverse transcription-polymerase chain reaction (RT-PCR) was done to further exclude RUNX1-RUNX1T1 rearrangement, although the likelihood of having undetected rearrangement by karyotyping and FISH studies is extremely low. What is the new aspect of your work? Here, we for the first time presented a case of AML with morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells, coinciding with CSF3R mutation, which is a rare mutation in AML while representing a potential novel therapeutic target. What is the central finding of your work? A rare case of CSF3R-mutated AML with no recurrent cytogenetic abnormality, showing unusual morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells. What is (or could be) the specific clinical relevance of your work? The rare morphological features present in this AML could potentially serve as a trigger to identify cases of AML with CSF3R mutation for targeted therapy. None. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. Not applicable. Not applicable. Not applicable. The data that support the findings of this study are available from the corresponding author upon reasonable request.