THP-1 Acute Myeloid Leukemia Cells Research Articles

Studying Signaling Pathway Activation in TRAIL-Resistant Macrophage-Like Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a malignant neoplasm characterized by extremely low curability and survival. The inflammatory microenvironment and maturation (differentiation) of AML cells induced by it contribute to the evasion of these cells from effectors of antitumor immunity. One of the key molecular effectors of immune surveillance, the cytokine TRAIL, is considered a promising platform for developing selective anticancer drugs. Previously, under in vitro conditions of the inflammatory microenvironment (a three-dimensional high-density culture of THP-1 AML cells), we demonstrated the emergence of differentiated macrophage-like THP-1ad clones resistant to TRAIL-induced death. In the present study, constitutive activation of proinflammatory signaling pathways, associated transcription factors, and increased expression of the anti-apoptotic BIRC3 gene were observed in TRAIL-resistant macrophage-like THP-1ad AML cells. For the first time, a bioinformatic analysis of the transcriptome revealed the main regulator, the IL1B gene, which triggers proinflammatory activation and induces resistance to TRAIL in THP-1ad macrophage-like cells.

C-5401331 identified as a novel T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) inhibitor to control acute myeloid leukemia (AML) cell proliferation.

T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a checkpoint protein expressed in exhausted T-cells during cancer scenarios. This exhaustion may end in T-cell effector dysfunction, resulting in suboptimal control of cancers like acute myeloid leukemia (AML). Use of immune checkpoint inhibitors (ICIs) to block checkpoint receptors such as Tim-3 is an emerging, revolutionary concept in the immuno-oncology therapeutic arena; however, ICIs are not effective on myeloid malignancies. Here, a multifaceted approach is utilized to identify novel compounds that target and inhibit Tim-3 with improved efficacy. High-throughput virtual screening of the ChemBridge small molecule library and molecular dynamics simulation yielded a lead molecule C-5401331 predicted to bind with high affinity and inhibit the activity of Tim-3. In vitro evaluations demonstrated the compound to have anti-proliferative effects on Tim-3-positive populations of THP-1 and HC-5401331 AML cells, inducing early and late phase apoptosis. With further development, the lead molecule identified in this work has potential to aid the natural "gatekeeper" functions of the body in immunocompromised AML cancer patients by successfully hampering the binding of Tim-3 to T-cells.

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy. Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

Casein Kinase II Inhibitor Sensitizes the Anti-Tumor Effect of EZH2 Inhibitor By Suppressing FOSL1 Expression in Acute Myeloid Leukemia

Background More effective combination therapy is emerging needed owing to the high mortality and poor outcome in acute myeloid leukemia (AML). The genomic amplification and overexpression of Enhancer of zeste homolog 2 (EZH2) occur in AML patients, and high expression of the Casein kinase 2 (CK2) is associated with poor survival in AML patients. Both EZH2 inhibitor and CK2 inhibitors are reported to inhibit cell proliferation and induce apoptosis in AML, however, the anti-leukemia effect of EZH2 inhibitor in combination with CK2 inhibitor has not been reported. The present study aims to investigate the effect of the combination of the EZH2 inhibitor DZNeP with CK2 inhibitor CX4945 in AML and possible underlying mechanisms. Methods A total of 40 newly diagnosed AML patients and 40 bone marrow control from healthy donors were obtained from our institute with the approval of the Ethics Committee. Cell Counting Kit-8 assay (CCK8), Cell cycle assay, and apoptosis assays were applied in THP-1 AML cells treated with control, DZNeP, CX4945, and CX4945+DZNeP. Global transcriptome analysis by RNA-seq was performed in U937 cells treated with 2μM DZNeP; 12.5μM CX4945 or vehicle for 48 hours. FOSL1 mRNA level was examined by qPCR; R2 genomics analysis and visualization application were used for comparison of the expression difference and the Kaplan-Meier overall survival analysis was conducted by the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Results DZNeP only has little effect on cell proliferation arrest in THP-1 cells, but it significantly sensitized the effect of CX4945, and CalcuSyn analysis showed the synergistic effect of the CX4945+DZNeP (Fig.1A). The treatment of CX4945+DZNeP combination induced the G0/G1 phase arrest in THP-1 cells versus single-drug control (Fig. 1B). The combination also significantly increased the % of the apoptotic cells in THP-1 cells as well as the AML-5b primary cells from the patient compared to single-drug controls (p<0.0001) (Fig.1C). These data indicate the synergistic anti-tumor effect of CX4945+DZNep combination in AML. To understand the molecular mechanism underlying the synergy, the global transcriptome was identified in U937 cells. The significantly different expression genes (DEGs) were identified upon CX4945 treatment (∣log2FC∣>1.5, FDR<0.05), or DZNeP treatment (∣log2FC∣>1.0, FDR<0.05) versus vehicle-control; and 1908 overlapped DEGs are identified upon either drug treatment, in which 596 were intersected (429 genes changed in the same direction but 167 changed in the opposite (Fig. 1D). KEGG enrichment analyses showed the overlapped DEGs upon the drug treatment mainly involved in cell cycle, DNA replication, and MAPK signaling pathway. (Fig.1E). It is worth noting that FOSL1 is dramatically downregulated in U937 cells upon treatment of either DZNeP or CX4945 (Fig1.D); and qPCR data showed that the mRNA level of FOSL1 is significantly downregulated by CX4945+DZNeP, DZNeP or CX4945 compared to the vehicle control (p<0.0001) in THP-1 cells (Fig. 1F). These data indicated that the combination exerts its anti-tumor effect by down-regulation of FOSL1 expression in AML. FOSL1 is a key component of the AP-1 transcriptional complex, and overexpression of FOSL1 is associated with invasion, migration, proliferation, and stemness in human solid cancers. We also examined the FOSL1 mRNA level in 40 newly diagnosed patients with AML versus 40 normal bone marrow controls. Results showed the mRNA level of FOSL1 is significantly higher in AML patients than that of normal controls (p<0.001) (Fig1.G). Metadata analysis showed that FOSL1 high expression is associated with short overall survival (p=0.033) (Fig.1H). These results not only suggested the oncogenic role of FOSL1 in AML but also further supported the CX4945+DZNeP combination-induced anti-tumor effect mediated by suppressing FOSL1 expression in AML. The mechanism model is summarized in Fig. 1E. Conclusion The combination of EZH2 inhibitor DZNeP and CK2 inhibitor CX4945 exerts a potent synergistic effect on proliferation arrest, cell cycle arrest, and apoptosis in AML by suppressing FOSL1 expression. Our results reveal the oncogenic role of FOSL1 in AML. Our data also provide experimental hints for the potential clinical trial of the novel combination in AML patients.

Vitamin D Receptor Antagonist MeTC7 Inhibits PD-L1.

Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.

Therapeutic Targeting of EP300/CBP By Bromodomain Inhibition in Acute Myeloid Leukemia

CCS1477 (also known as inobrodib) is a potent and selective inhibitor of the bromodomains of EP300 and CBP, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. In pre-clinical analyses we found that CCS1477 induced dose-dependent growth inhibition of a wide range of acute myeloid leukemia (AML) cell lines and primary patient samples of diverse molecular subtypes at low nanomolar concentrations. Growth inhibition was predominantly due to cell cycle arrest and differentiation; there was only modest apoptosis. In vivo, in contrast to control mice which all developed short latency AML, all animals with experimentally initiated MLL-AF9 and MLL-AF10 driven AMLs (in a retroviral transduction and transplantation syngeneic model) treated with 30mg/kg daily PO CCS1477 survived to termination of the experiment at 220 days. In subcutaneous xenograft models, there was dose-dependent growth inhibition of MOLM16 AML cell tumours, with 20mg/kg daily for 21 days completely suppressing tumour growth and no tumour regrowth until 14 days following the end of treatment. Mechanistically, we found that 100nM CCS1477 induced rapid and selective depletion of EP300 in particular from MYB binding sites in THP1 AML cells. ChIPseq demonstrated that in steady state the strongest recruitment of EP300 to chromatin in THP1 AML cells was at sites of MYB and CEBPA transcription factor occupancy, although EP300 was also found genome-wide at sites occupied by other factors including RUNX1 and ETS. Following CCS1477 treatment there was, within six hours, selective loss of EP300 at MYB occupied sites, co-localized reduced chromatin accessibility and histone H3K27 acetylation, and stalling of RNA polymerase at subordinate genes with reduced transcription. This was associated with a dose-dependent redistribution of EP300 to alternate sites and up regulation of differentiation genes. In our ongoing phase I/IIa clinical trial investigating the safety and efficacy of CCS1477 in advanced haematological malignancies (NCT04068597), we have observed therapeutically beneficial granulocytic differentiation responses in patients with high-risk myelodysplasia or relapsed AML. For example, a patient with myelodysplasia with excess blasts 2 (MDS-EB2) evolving to AML who had received three cycles of azacitidine without response was treated with CCS1477 50mg PO (once daily, three days/week). Pre-CCS1477 treatment there was profound neutropenia and multiple admissions for sepsis. After CCS1477 treatment the neutrophil count rapidly normalized and, despite a brief admission for splenic infarction in the first week, the patient remained well for 5 months with no infective episodes. Treatment was stopped due to a rising leukocyte count. Similar responses were seen in other patients. In one of these, single cell RNA sequencing of serial blood and bone marrow samples confirmed appearance of mature myeloid cells on CCS1477 treatment and, within the blast population, up regulation of a multilineage differentiation programme and down regulation of a stem cell programme. Reflecting our findings in cell line models, there was also up regulation of genes induced by MYB knockdown and concordant up and down regulation of genes regulated by CEBPA in THP1 AML cells. Finally, we evaluated the activity of CCS1477 in xenograft models in combination with existing standard of care agents. Azacitidine and CCS1477 in particular demonstrated synergistic growth inhibitory activity in the MOLM16 model, as did venetoclax in MOLM16 and MV(4;11) models. These encouraging pre-clinical and early phase clinical findings set the stage for the clinical trial evaluation of CCS1477 in combination with azacitidine and venetoclax.

Pyrimethamine, a STAT3 Inhibitor, Synergizes with Venetoclax and Shows Efficacy in Hypomethylating Agent Resistant MDS and AML

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arise after accumulation of multiple genetic and epigenetic alterations in hematopoietic stem cells (HSCs). Alterations in HSCs promote malignant transformation and give rise to leukemic stem cells (LSCs). Current therapies for MDS and AML, including recently FDA approved agents such as venetoclax (Ven), a Bcl-2 inhibitor, have failed to eliminate LSCs, therefore, molecular targeting of aberrant LSCs may be a potential strategy for developing targeted drugs capable of eliminating LSCs. Studies have shown that aberrant HSCs exhibit multiple disrupted cellular pathways, including overexpression of the signal transducer and activator of transcription 3 (STAT3) (Shastri, 2017). Elevated levels of STAT3 are observed in MDS and AML patient samples. Interestingly, an increased level of STAT3 in MDS and AML patients is predictive of a significantly adverse prognosis, thus supporting a critical role of STAT3 signaling in MDS and AML. We have previously shown that an antisense oligonucleotide inhibitor of STAT3, gets rapidly incorporated into HSCs and induces apoptosis and downregulation of STAT3 when compared to health controls (Shastri, JCI 2018). An initial screen to identify compounds that could target STAT3 revealed that pyrimethamine (PYR), an FDA approved anti-parasitic drug, inhibits STAT3 transcriptional activity at concentrations readily achieved in patients (Nelson, 2011). Therefore, we are further evaluating the efficacy of PYR for treatment of MDS and AML. HMA and Ven resistance is a critical issue in MDS/AML and can occur due to the persistence of relapse associated aberrant stem and progenitor cells that overexpress STAT3. Hence, we are evaluating the efficacy of PYR in a hypomethylating agent (HMA) and venetoclax (Ven) resistant model. Our in vitro studies show that PYR can inhibit viability by inducing apoptosis in multiple leukemic cells within its therapeutic index. Western blot analysis reveals that STAT3 protein is overexpressed in a Ven resistant MOLM13 AML line, and in a HMA resistant cell THP1 AML cell line. In HMA and Ven resistant leukemia cell lines, PYR can significantly decrease proliferation when combined with azacytidine (P= 0.0109) (Fig 1A) or venetoclax (P= 0.0271) treatments. Remarkably, PYR and Ven drug combination show a strong synergistic interaction in HMA resistant cell line (Z score = 14.523) and Ven resistant cell line (Z score = 11.065). An in vivo study to evaluate the efficacy of PYR shows that PYR treatment leads to a significant decrease of blasts counts in a cell-line derived xenograft (CDX) leukemic mouse model (Fig 1B) compared to a PBS control. In addition, primary MDS patient samples treated with PYR show increased myeloid differentiation compared to healthy controls. Our results show that MDS/AML cell lines, including HMA and Ven resistant leukemia cell lines, are sensitive to PYR treatment, in a dose dependent manner within the therapeutic index of STAT3 inhibition (5-10 uM). Interestingly, PYR with Ven drug combination shows significant reduction in cell proliferation and likely synergy. Our current findings support PYR synergy with Ven and the combination as a possible therapeutic option for patients with MDS/AML after prior treatment with hypomethylating agents for MDS and AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SGOL1-AS1 enhances cell survival in acute myeloid leukemia by maintaining pro-inflammatory signaling

Epigenetic dysregulation is a key feature of most acute myeloid leukemia (AML). Recently, it has become clear that long noncoding RNAs (lncRNAs) can play a key role in epigenetic regulation, and consequently also dysregulation. Currently, our understanding of the requirements and roles of lncRNAs in AML is still limited. Here, using CRISPRi screening, we identified the lncRNA SGOL1-AS1 as an essential regulator of survival in THP-1 AML cells. We demonstrated that SGOL1-AS1 interacts with chromatin-modifying proteins involved in gene repression and that SGOL1-AS1 knockdown is associated with increased heterochromatin formation. We also observed that loss of SGOLl-AS1 results in increased apoptosis and the downregulation of pro-inflammatory genes. In AML patients, high expression of SGOL1-AS1 correlates with both pro-inflammatory gene expression and poor survival. Altogether, our data reveal that SGOL1-AS1 is an essential regulator of cell survival in AML cell lines and a possible regulator of pro-inflammatory signaling in AML patients.

RAB3D/MDM2/β-catenin/c-MYC axis exacerbates the malignant behaviors of acute myeloid leukemia cells in vitro and in vivo.

RAB3D, a small Ras-like GTPase involved in regulating secretory pathway, plays a cancer-promoting role in several solid tumors. However, its role in leukemogenesis remains unknown yet. Acute myeloid leukemia (AML) is a common acute leukemia with a high mortality. Here, we found the higher expression of RAB3D in bone marrow mononuclear cells derived from AML patients (n = 54) versus healthy participants (n = 20). The following loss- and gain-of-function experiments demonstrated that RAB3D promoted growth, enhanced colony formation and accelerated G1/S transition of U937, THP-1 and KG-1 AML cells. RAB3D silencing inhibited tumorigenesis of AML cells in vivo and delayed AML cells-induced death of mice. Interestingly, the expression of RAB3D is positively correlated with that of an oncogene mouse double minute 2 (MDM2) in bone marrow mononuclear cells of AML patients (r = 0.923, p < 0.001). Intracellular MDM2 was conjugated with more ubiquitins and degraded faster when RAB3D was silenced. A commonly therapeutic target of AML, β-catenin signaling, was activated by RAB3D overexpression, but deactivated after MDM2 was silenced. The RAB3D-induced proliferation acceleration and β-catenin activation were abolished by MDM2 knockdown, implying that RAB3D function by stabilizing MDM2. In addition, c-MYC, a β-catenin downstream effector, was recruited directly to the RAB3D gene promoter (-360/-349 and -136/-125 sites) and induced its transcription. Collectively, this study demonstrates that RAB3D may exacerbate the malignant behaviors of AML cells through forming a positive feedback loop with MDM2/β-catenin/c-MYC signaling. RAB3D might be a novel target of clinical AML treatment.

A Novel BRD Family PROTAC Inhibitor dBET1 Exerts Great Anti-Cancer Effects by Targeting c-MYC in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis remains dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of patients with AML. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a proteolysis-targeting chimera (PROTAC) against the bromodomain and extra-terminal domain (BET) family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment arrested cell cycling and enhanced apoptosis and c-MYC was identified as the downstream target. Collectively, our results indicated that dBET1 had broad anti-cancer effects on AML cell lines with different molecular lesions and provided more benefits to patients with AML.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

CD123 and CD33 Co-Targeting By Balanced Signaling on CAR-CIK Cells Reduces Potential Off-Target Toxicity While Preserving the Anti-Leukemic Activity of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse . Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3) full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells.Fresh and frozen peripheral blood mononuclear cells were transduced with retroviral vectors during the CIK cell differentiation process . The functional activity of CAR-CIK cells was assessed in vitro by means of short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines (THP-1 and KG-1) or with a CD123 positive human endothelial cell line (TIME). To assess the myelotoxicity against HSPC, CD34+ cells were sorted after 24h co-culture with CAR-CIK cells and mixed with methylcellulose-based semisolid medium to evaluate Colony Forming Units (CFU) after 14 days through an automated imaging and counting system for hematopoietic colonies (Stemvision). The in vivo anti-leukemic activity was evaluated by monitoring luciferase-expressing KG1 AML cell line growth over time in NSG mice treated with 3 infusions of all different CAR-CIK conditions.Dual CAR-CIK cells (IL-3z.CAR/CD33.CCR) display a potent and specific in vitro anti-leukemic efficacy against all the AML cell lines tested, compared to single targeting IL-3z.CAR and non-transduced CIK cells. To further minimize toxicities against healthy cells exploiting differences in CD123 antigen density between leukemic and healthy cells, we generated a low affinity dual CAR decreasing the IL3z.CAR binding affinity to CD123, by site-directed mutagenesis. Low affinity dual CAR-CIK cells show irrelevant cytotoxicity against the TIME endothelial cell line, comparable to non-transduced CIK cells, while preserving the efficacy against THP1 and KG1 AML cell lines. As a confirm of the reduced reactivity against endothelial cells, low affinity dual CAR-CIK cells produce the same levels of IL-2 and IFNγ either alone or in the presence of TIME cell line. Low affinity dual CAR-CIK cells also preserve the in vitro CFU-E, CFU-GM and CFU-GEMM colony forming capacity as compared to wild type (wt) dual CAR-CIK and wt IL-3z.CAR-CIK cells, highlighting the possibility to find a therapeutic window to minimize toxicity on healthy cells. The anti-leukemic activity of low affinity dual CAR-CIK cells has been confirmed also in vivo, with a significant suppression of leukemic growth against KG1 AML cell line. Mice treated with 3 infusions of low affinity dual CAR-CIK cells exhibited a significant better anti-leukemic profile, when compared to IL-3-single targeting CAR-CIK cells in terms of tumor growth and overall survival.These preclinical data demonstrate a powerful antitumor efficacy mediated by low affinity dual targeting IL-3z.CAR/CD33.CCR CIK cells against AML targets without any relevant in vitro toxicity on HSPC and endothelial cells, offering a proof-of-concept strategy to increase selectivity for CD123+/CD33+ AML cells whilst reducing the risk of “on-target off-tumor toxicity”. DisclosuresPerriello: Novartis: Other: Advisory Board. Martelli: Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals: Honoraria. Falini: Rasna Therapeutics: Honoraria. Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria.

CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1

CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

Inhibition of XPO1 By KPT-330 (Selinexor) Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor ABT-199 (Venetoclax) through Downregulation of Mcl-1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a serious disease and despite an improved understanding of the biology of AML, little has changed in the treatment of the disease over the last four decades. The 5 year survival rates of AML remain frustratingly low. Resistance to frontline chemotherapy remains the main cause of treatment failure; therefore a new treatment modality is urgently needed.Bcl-2 is a promising therapeutic target; it is overexpressed in both bulk AML cells and leukemia-initiating cells. The Bcl-2-selective inhibitor ABT-199 (Venetoclax) shows promising anti-leukemic activity in patients with high-risk relapsed/refractory AML or unfit for intensive chemotherapy. However, resistance through another Bcl-2 family protein, Mcl-1, was shown by our lab and others to limit the anti-leukemic activity of ABT-199. Previous studies from our lab have shown that ABT-199 monotherapy decreases the association of Bim with Bcl-2 but is compensated for by its increased association with Mcl-1. We also found that ABT-199 increases cellular Mcl-1 levels by extending the protein's half-life. Based on these previous studies we tested the efficacy of direct inhibition of Bcl-2 by ABT-199 and Mcl-1 by A-1210477. ABT-199 and A-1210477 synergistically induced apoptosis in both ABT-199-resistant and sensitive AML cells. This combination treatment disrupted binding of Bim with Bcl-2 and Mcl-1 concurrently, with no observed binding of Bim to Bcl-xL. This work has demonstrated that targeting both Bcl-2 and Mcl-1 is necessary for AML treatment. However, our work also indicated that direct targeting of Mcl-1 with A-1210477 to enhance the anti-leukemic activity of ABT-199 might cause increased toxicity. Thus, indirect targeting of Mcl-1 may represent a better approach to enhance the anti-leukemic activity of ABT-199 in AML.It was recently reported that targeting exportin 1 (XPO1), a nuclear exporter overexpressed in AML cells, selectively targets leukemia-initiating cells and can decrease Mcl-1 levels in cancer cells, though the underlying mechanism is unknown. KPT-330 (Selinexor) is a XPO1-selective inhibitor which is currently being tested in clinical trials for AML. Preliminary results have shown that KPT-330 induces a response at well-tolerated doses in AML and other hematological malignancies. Thus, XPO1 inhibition may represent a viable strategy for downregulating Mcl-1 to synergize with ABT-199 treatment in AML.To test our hypothesis we tested various concentrations of ABT-199 and KPT-330 alone and in combination in AML cell lines and primary patient samples. At 24 h, synergistic induction of apoptosis was observed between the two drugs in THP-1, CTS, Molm-13, and OCI-AML3 AML cell lines. Synergy was also found in 4 relapsed and 6 newly diagnosed primary AML patient samples. To test the effect of drug treatment on leukemia-initiating cells, we performed colony formation assays on primary AML patient samples post drug treatment and found that combination treatment significantly enhanced inhibition of colony formation when compared to single drug treatment, demonstrating that KPT-330 combined with ABT-199 is effective in targeting the leukemic bulk and the leukemia-initiating cells.To determine the mechanism by which the combination works, we measured the protein levels of various Bcl-2 family members. Decreased protein levels for Mcl-1 and surprisingly Bim were observed post KPT-330 or KPT-330 plus ABT-199 treatment, while protein levels for Bcl-2 and Bcl-xL remained unchanged. A time course experiment revealed that Mcl-1 levels decreased at apoptosis initiation while Bim levels decreased after apoptosis initiation, suggesting that Mcl-1 plays a critical role in apoptosis induced by the drug combination, while Bim may not. Mcl-1 knockdown enhanced combination treatment of ABT-199 and KPT-330. Reciprocal Mcl-1 overexpression partially rescued the cells from drug treatment but was incomplete, likely due to Mcl-1 overexpression not being high enough and/or non-Mcl-1 pathways through which KPT-330 acts. In summary, Mcl-1 is modulated by KPT-330 treatment alone and in combination with ABT-199 and this modulation of Mcl-1 plays an important role in initiation of apoptosis in response to treatment. Additional studies are underway to further elucidate the molecular mechanisms of combination ABT-199 and KPT-330 treatment and to determine the in vivo anti-leukemic efficacy in NSG mouse AML models. DisclosuresNo relevant conflicts of interest to declare.

Genome-scale integration of transcriptome and metabolome unveils squalene synthase and dihydrofolate reductase as targets against AML cells resistant to chemotherapy.

The development of resistance to chemotherapeutic agents, such as Doxorubicin (DOX) and cytarabine (AraC), is one of the greatest challenges to the successful treatment of Acute Myeloid Leukemia (AML). Such acquisition is often underlined by a metabolic reprogramming that can provide a therapeutic opportunity, as it can lead to the emergence of vulnerabilities and dependencies to be exploited as targets against the resistant cells. In this regard, genome-scale metabolic models (GSMMs) have emerged as powerful tools to integrate multiple layers of data to build cancer-specific models and identify putative metabolic vulnerabilities. Here, we use genome-scale metabolic modelling to reconstruct a GSMM of the THP1 AML cell line and two derivative cell lines, one with acquired resistance to AraC and the second with acquired resistance to DOX. We also explore how, adding to the transcriptomic layer, the metabolomic layer enhances the selectivity of the resulting condition specific reconstructions. The resulting models enabled us to identify and experimentally validate that drug-resistant THP1 cells are sensitive to the FDA-approved antifolate methotrexate. Moreover, we discovered and validated that the resistant cell lines could be selectively targeted by inhibiting squalene synthase, providing a new and promising strategy to directly inhibit cholesterol synthesis in AML drug resistant cells.

Resveratrol ve Seramid Metabolizması Enzim İnhibitörleri İle Kombinasyonlarının FLT3 Pozitif Akut Miyeloid Lösemideki Sitotoksik Etkileri

Sphingolipids determine the cell fate by regulating cell proliferation and growth. Ceramide, growth inhibitory lipid, might be produced through de novo pathway or salvage pathway, which is converted to proliferation inducers sphingosine-1-phosphate (S1P) and glucosyl ceramide (GC) by sphingosine kinase (SK) and glucosyl ceramide synthase (GCS), respectively. It is aimed to investigate therapeutic potential of resveratrol on FLT3 overexpressing acute myeloid leukemia (AML) cells by pharmacological targeting of ceramide metabolism. The cytotoxic effects of resveratrol, SK inhibitor (SKI II), GCS inhibitor (PDMP) and the combinations of resveratrol with SK-1 inhibitor and GCS inhibitor on THP-1 and OCI-AML3 FLT3 overexpressing AML cells were investigated by MTT cell viability assay in a time- and concentration-dependent manner. Apoptotic effect of resveratrol was analyzed by annexin V/PI double staining using flow cytometry. Resveratrol decreased cell viability and induced apoptosis in both cell lines (p&amp;lt;0.05 considered significant). There were synergistic cytotoxic effects of resveratrol with co-administration of SK-1 inhibitor and GCS inhibitor at 48 h (p&amp;lt;0.05 considered significant). This preliminary data showed for the first time that resveratrol might inhibit the viability of FLT3 overexpressing AML cells through targeting ceramide metabolism and inducing apoptosis, which needs to be further clarified mechanistically.

Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer

BackgroundResistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML.MethodsThe human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML.ResultsProlonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied.ConclusionsDespite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.

Venetoclax Inhibition of Pyrimidine Synthesis Guides Methods for Integration with Decitabine or 5-Azacytidine That Are Non-Myelosuppressive

Venetoclax (Ven) administered daily with pulse-cycled parenteral decitabine (Dec) or 5-azacytidine (5Aza) is standard therapy for acute myeloid leukemia (AML) in the elderly. In practice, toxicity/myelosuppression is frequent, and prompts Ven dose reductions, but by guess-work, because the mechanism downstream of BCL2-inhibition by which Ven augments Dec/5Aza activity is unclear. For the first time, we show that Ven inhibits de novo pyrimidine synthesis, an effect that can guide its integration with Dec/5Aza in a way that enhances anti-AML activity without suppressing normal myelopoiesis. Dec and 5Aza are pro-drugs processed by pyrimidine metabolism into a deoxycytidine analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1), a pharmacodynamic effect that terminates malignant but not normal self-replication. We recently demonstrated that Dec- and 5Aza-resistance emerges automatically from adaptive responses of the pyrimidine metabolism network to Dec/5Aza-induced nucleotide perturbations, such that Dec/5Aza processing into DNMT1-depleting nucleotide is forestalled (Leukemia - https://rdcu.be/b58pS). A key element in this auto-resistance is upregulated de novo pyrimidine synthesis, that out-competes salvaged Dec/5Aza. De novo pyrimidine synthesis has an electron-transport dependent mitochondrial step executed by dihydroorotate dehydrogenase (DHODH): BCL2-inhibition by Ven depolarizes mitochondrial membranes - we examined for the first time Ven impact on DHODH/pyrimidine synthesis (others have focused on apoptosis and other metabolic consequences of mitochondrial depolarization). Consistent with Ven inhibiting DHODH/pyrimidine synthesis, both Ven and the direct DHODH inhibitor teriflunomide, at non-apoptotic concentrations, significantly decreased cytidine- and deoxycytidine triphosphate (CTP, dCTP) in AML cells (Fig1A). To see if this effect of Ven can counter Dec/5Aza resistance, we selected for THP1 AML cells double-resistant to Dec 0.5 μM and 5Aza 5 μM - these cells upregulated expression of de novo pyrimidine synthesis enzymes and contained uridine, cytidine and deoxycytidine at levels up to 7-fold higher than parental cells. The double-resistant AML cells were significantly cytoreduced by Ven at a clinically relevant concentration of 1 μM, even though parental THP1 AML cells were minimally sensitive (Fig1B). Consistent with inhibition of de novo pyrimidine synthesis as the mechanism, this action was significantly abrogated by cytidine supplementation (Fig1B). We previously showed that timed alternation of Dec with 5Aza, and incorporation of the cytidine deaminase inhibitor tetrahydrouridine (THU), counters metabolic Dec or 5Aza-resistance to extend non-cytotoxic DNMT1-depletion and survival in vivo (https://rdcu.be/b58pS). However, AML still eventually progresses, via upregulated de novo pyrimidine synthesis. In considering use of Ven to counter this mode of resistance, we reasoned that concurrent administration risks antagonism, because Ven can cause transient cytostasis, and DNMT1-depletion by Dec/5Aza is S-phase dependent. Prior Ven administration, however, creates effect-time sufficient to deplete endogenous nucleotides from AML cells and hence upregulate pyrimidine salvage (that uptakes Dec/5Aza) (Fig1A), as well as resumed cell cycle. Therefore, in vivo in a patient-derived xenotransplant model (PDX) of AML, we introduced Ven (at human equivalent dose), parsimoniously 2X/week, the day before each Dec or 5Aza administration, and at the time of overt AML progression on the base THU-Dec/5Aza regimen. This minimalist Ven application significantly extended survival (time-to-distress) even though it was initiated at progression (Fig1C). As expected, use of this regimen upfront in a PDX of Dec/5Aza-resistant AML produced even greater (several-fold) survival extension (treatment ongoing) vs the base regimen (Fig1D). A non-cytotoxic/non-myelosuppressive mechanism-of-action was confirmed by serial blood counts on-therapy. In practice, Ven dose in combination with Dec or 5Aza to treat AML is frequently empirically reduced because of toxicity/myelosuppression. Instead, Ven inhibition of de novo pyrimidine synthesis can guide mechanism-based, intermittent administration that avoids toxicity/myelosuppression yet enhances Dec/5Aza anti-AML activity. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago.