Mouse Acute Myeloid Leukemia Cells Research Articles

Antineoplastic Activity of Sodium Caseinate in a Cytarabine-Resistant Mouse Acute Myeloid Leukemia Cell Line.

Acute myeloid leukemia (AML) is a hematological neoplasm of rapid and progressive onset, and is the most common form of leukemia in adults. Chemoresistance to conventional treatments such as cytarabine (Ara-C) and daunorubicin is a main cause of relapse, recurrence, metastasis, and high mortality in AML patients. It is known that sodium caseinate (SC), a salt derived from casein, a milk protein, inhibits growth and induces apoptosis in acute myeloid leukemia cells but not in normal hematopoietic cells. However, it is unknown whether SC retains its antileukemic effect in cytarabine-resistant AML cell lines. To evaluate the antineoplastic effect of SC in cytarabine-resistant leukemia models. The SC inhibits the growth and induces apoptosis in parental WEHI-3 AML cells. Here, we generated two cytarabine-resistant sublines, WEHI-CR25 and WEHI-CR50, which exhibit 6- and 16-fold increased resistance to cytarabine, respectively, compared to the parental WEHI-3 cells. Thus, these sublines mimic a chemoresistant model. We demonstrate that WEHI-CR25 and WEHI-CR50 cells retain sensitivity to SC, similar to parental WEHI-3 cells. This sensitivity results in inhibited cell proliferation, induced apoptosis, and increased expression of ENT1 and dCK, molecules involved in the entry and metabolism of Ara-C, while decreasing MDR1 expression. Additionally, we observed that SC prolonged the survival of WEHI-CR50 tumor-bearing mice, despite their resistance to Ara-C. This is the first evidence that SC, a milk protein, may inhibit proliferation and induce apoptosis in cytarabine-resistant cells.

Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation invivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing invivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, Tcell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.

Inhibiting AGTR1 reduces AML burden and protects the heart from cardiotoxicity in mouse models.

Clinical treatment of acute myeloid leukemia (AML) largely relies on intensive chemotherapy. However, the application of chemotherapy is often hindered by cardiotoxicity. Patient sequence data revealed that angiotensin II receptor type 1 (AGTR1) is a shared target between AML and cardiovascular disease (CVD). We found that inhibiting AGTR1 sensitized AML to chemotherapy and protected the heart against chemotherapy-induced cardiotoxicity in a human AML cell-transplanted mouse model. These effects were regulated by the AGTR1-Notch1 axis in AML cells and cardiomyocytes from mice. In mouse cardiomyocytes, AGTR1 was hyperactivated by AML and chemotherapy. AML leukemogenesis increased the expression of the angiotensin-converting enzyme and led to increased production of angiotensin II, the ligand of AGTR1, in an MLL-AF9-driven AML mouse model. In this model, the AGTR1-Notch1 axis regulated a variety of genes involved with cell stemness and chemotherapy resistance. AML cell stemness was reduced after Agtr1a deletion in the mouse AML cell transplant model. Mechanistically, Agtr1a deletion decreased γ-secretase formation, which is required for transmembrane Notch1 cleavage and release of the Notch1 intracellular domain into the nucleus. Using multiomics, we identified AGTR1-Notch1 signaling downstream genes and found decreased binding between these gene sequences with Notch1 and chromatin enhancers, as well as increased binding with silencers. These findings describe an AML/CVD association that may be used to improve AML treatment.

Nanoparticle-encapsulated retinoic acid for the modulation of bone marrow hematopoietic stem cell niche

More effective approaches are needed in the treatment of blood cancers, in particular acute myeloid leukemia (AML), that are able to eliminate resistant leukemia stem cells (LSCs) at the bone marrow (BM), after a chemotherapy session, and then enhance hematopoietic stem cell (HSC) engraftment for the re-establishment of the HSC compartment. Here, we investigate whether light-activatable nanoparticles (NPs) encapsulating all-trans-retinoic acid (RA+NPs) could solve both problems. Our in vitro results show that mouse AML cells transfected with RA+NPs differentiate towards antitumoral M1 macrophages through RIG.1 and OASL gene expression. Our in vivo results further show that mouse AML cells transfected with RA+NPs home at the BM after transplantation in an AML mouse model. The photo-disassembly of the NPs within the grafted cells by a blue laser enables their differentiation towards a macrophage lineage. This macrophage activation seems to have systemic anti-leukemic effect within the BM, with a significant reduction of leukemic cells in all BM compartments, of animals treated with RA+NPs, when compared with animals treated with empty NPs. In a separate group of experiments, we show for the first time that normal HSCs transfected with RA+NPs show superior engraftment at the BM niche than cells without treatment or treated with empty NPs. This is the first time that the activity of RA is tested in terms of long-term hematopoietic reconstitution after transplant using an in situ activation approach without any exogenous priming or genetic conditioning of the transplanted cells. Overall, the approach documented here has the potential to improve consolidation therapy in AML since it allows a dual intervention in the BM niche: to tackle resistant leukemia and improve HSC engraftment at the same time.

A gain-of-function p53 mutant synergizes with oncogenic NRAS to promote acute myeloid leukemia in mice.

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in NrasG12D/+ p53-/- (NP-/-) mice, NrasG12D/+ p53R172H/+ (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP-/- cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB-inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.

The XPO7/Npat Axis Inactivation Is a Therapeutic Vulnerability for TP53-Mutated AML

In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.

Primary U2AF1 S34F Mutated Hematopoietic Cells Are Sensitive to Nonsense-Mediated RNA Decay Disruption In Vivo

Expression of mutant splicing factors alters RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells, and the differential sensitivity is partially dependent on R-loop formation. However, the impact of NMD disruption on the viability of primary hematopoietic cells with splicing factor mutations has not been tested in vivo. We engineered mouse MLL-AF9 acute myeloid leukemia (AML) cells to inducibly express a wild type ( U2AF1 WT) or mutant ( U2AF1 S34F) transgene of the U2AF1 splicing factor, and tested the effects of inhibiting the NMD pathway protein kinase, SMG1, in vivo. We injected U2AF1 WT and U2AF1 S34F mouse AML cells (B6, CD45.2) into congenic, irradiated secondary recipient mice (B6, CD45.1), induced transgene expression, and then treated the mice for 2 weeks with either a SMG1 inhibitor (SMG1i) at 60 mg/kg/day or vehicle. SMG1i treatment increased the overall survival (OS) of mice bearing U2AF1S34F AML cells (median OS of 53 days compared to 42 days for mice treated with vehicle; p =0.0003), but did not impact the OS of mice with U2AF1WT AML cells (median OS of 33 days for SMG1i-treated mice compared to median OS of 32 days for vehicle-treated mice, p=0.360). We extended these findings to a second cohort of mice, where we transplanted one primary U2AF1 S34F banked mouse AML sample into 48 secondary recipient mice. To allow direct comparison of isogenic cells with or without U2AF1 S34F expression, half the mice were treated with doxycycline to induce U2AF1 S34F expression, and half of the mice did not receive doxycycline. SMG1i treatment increased the OS of mice expressing the U2AF1S34F mutant allele (median OS of 66 days, compared to median OS of 54 days for mice treated with vehicle; p<0.0001), but had no impact on survival of mice bearing AML cells where U2AF1 S34F was not expressed (median OS of 50 days, compared to median OS of 52 days for mice treated with vehicle; p=0.95). To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we first examined the effects of SMG1i treatment on cell cycle progression and cell death in primary cells. We observed that primary U2AF1 S34F-expressing AML cells treated with SMG1i ex vivo (24 hours) had an increased fraction of cells in G2/M phase compared to U2AF1 WT-treated cells (mean 48% vs. 11% at 100nM, respectively, p<0.001). In addition, the fraction of Annexin V + cells was increased in U2AF1 S34F-expressing cells treated with SMG1i ex vivo compared to U2AF1 WT treated cells (mean fold-change at 500nM relative to DMSO 4.0 vs. 2.3, respectively, p=0.027). We then isolated primary mouse AML cells after 7 days of in vivo SMG1i treatment and analyzed R-loops (S9.6 antibody) and DNA damage (gH2AX) by immunofluorescence. SMG1i-treated U2AF1 S34F cells had a marked increase in R-loop levels compared to U2AF1 WT-treated cells (mean S9.6 nuclear intensity, 306.9 vs 182.6, respectively, p<0.0001). In addition, H2AX phosphorylation was increased in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (mean gH2AX nuclear intensity, 136.7 vs 87.8, respectively p<0.0001).Thus, disrupting NMD in splicing factor mutant AML cells alters cell cycle progression and causes R-loop accumulation, DNA damage, and apoptotic cell death. The NMD pathway has been implicated in the regulation of the unfolded protein response (UPR) that controls protein homeostasis and impacts cell viability. We generated K562 cell lines expressing doxycycline-inducible U2AF1 S34F or U2AF1 WT constructs and confirmed increased sensitivity of U2AF1 S34F cells treated with SMG1i(IC50 128.1nM vs 578.5nM at 72 hours, respectively, p<0.0001). Next, we performed RNA-sequencing on U2AF1 WT and U2AF1 S34F cells that were treated with vehicle or SMG1i for 24 hours (n=3) and observed upregulation of endoplasmic reticulum and UPR pathway genes in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (GSEA; FDR<0.01). Validation of these findings using primary mouse AML cells is ongoing, including the analysis of key regulators and protein biomarkers of the UPR pathway. The vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility for therapeutic targeting of NMD to treat myeloid malignancies with aberrant splicing.

Local Oxygen Dictates Hematopoietic Cell Growth and Function

Functional hematopoietic stem (HSC) and progenitor cells (HPC) are responsible for the replenishment of blood/immune cells, are a source of treatment for diseases, and when dysregulated are a source of hematological neoplasia. To identify factors that impact HSC/HPC functional competency, we performed a transcriptomic screen which revealed that human cord blood (CB) HSCs/HPCs capable of early engraftment in an NSG mouse model of transplantation exhibited enriched expression of gene programs that vary with changes in oxygen (O 2) availability compared to non-transplanted input cells. This is physiologically and clinically relevant as HSCs/HPCs and diseased hematopoietic cells such as acute myeloid leukemia (AML) are exposed to O 2 tensions ranging from <1% to 21% as they function in various anatomical sites or are harvested for clinical applications. Thus, there are translational and practical rationales for understanding the growth and functional properties that hematopoietic cells exhibit across the range of O 2 they experience physiologically or artificially, which we examined here. First we found significantly different frequencies of HSCs/HPCs and colony forming units (CFU) in human bone marrow (BM), mobilized peripheral blood (PB), and CB, which have varied O 2 tensions, suggesting O 2 availability may impact hematopoiesis. To directly examine this, human CB CD34+ cells were split and plated in CFU assays or expanded in growth stimulating conditions at 1% O 2, 3% O 2, 5% O 2, 14% O 2 (physiologic tensions), or 21% O 2 (processing tension) . HPCs expanded best and formed more differentiated CFUs in high physiologic O 2 tensions and extra physiologic O 2 tensions, while HSCs expanded better and more potent CFUs were formed in a mid-physiologic O 2 tension (5%). This suggests that O 2 dependent pathways are critical to HSC/HPC growth but there may be a “sweet spot” for early HSCs/HPCs that allows for proliferation while maintaining self-renewal and potency to maintain a pool of primitive cells, possibly mirroring in vivo O 2 responses. In in vivo functional HSC/HPC analyses, NSG mice transplanted with CB units expanded in physiologic O 2 exhibited significantly better early neutrophil recovery and long-term myeloid reconstitution compared to input, suggesting that growth and maintenance of HSC/ myeloid HPC functional competency is better balanced in physiologic O 2 compared to extra physiologic tensions. Platelet recovery and lymphoid reconstitution were significantly better in mice receiving uncultured control CB, suggesting expansion does not affect all HSC/HPC subpopulations equally. Mechanistically, cells grown in low O 2 tensions (1-3%) had higher frequencies of early HSCs/HPCs, significantly lower intracellular ROS levels, a significantly higher percentage of cells in cell cycle G0, and lower levels of phosphorylated γH2AX than those grown in higher O 2. These data suggest that while to some extent HSCs/HPCs require O 2 to proliferate, they also exhibit properties associated with exhaustion when maintained in higher O 2. We next examined the effects varying O 2 tensions have on diseased hematopoietic cells using primary mouse AML cells or oncogene transformed human CB CD34+ cell lines. AML cells proliferated significantly more rapidly and formed more CFUs in higher O 2 tensions, suggesting that O 2 sensing pathways may be a dependency in AML that could affect disease progression as local O 2 tensions become perturbed. AML cells grown in lower O 2 exhibited lower resting levels of phosphorylated γH2AX (measured by flow cytometry) and lower O 2 consumption rates (measured by Seahorse metabolic flux analyzer), suggesting they are less susceptible to cellular stress. In line with this, AML cells maintained in low O 2 exhibited significantly better proliferation and recovery after treatment with the chemotherapeutic cytarabine compared to those in higher O 2 tensions. Thus, diseased cells growing in lower O 2 niches may be less susceptible to treatment and therefore may be a source of chemoresistance or relapse. These data reveal important roles for O 2 in balancing hematopoietic cell growth with normal and diseased cell function. Manipulating O 2 tension or O 2 sensing pathways could be used to improve HSC/HPC expansion for transplantation or to treat disease. Transcriptomic studies of HSCs/HPCs and AML cells are ongoing to determine the O 2 sensing mechanisms that dictate cellular function.

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p<0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p<0.01).

Preclinical Evaluation of Bisantrene As Single Agent and in Combination with Decitabine for Acute Myeloid Leukemia

Anthracyclines, particularly doxorubicin and idarubicin, together with cytarabine, have comprised standard of care induction chemotherapy in acute myeloid leukemia (AML) for almost 4 decades. Although 60% of patients achieve remission, the majority relapse within 1-2 years. As such, outcomes in AML remain poor, with overall 5-year survival around 27%. In elderly AML patients the situation is worse, with 80% dying within one year of diagnosis. Toxicity of induction therapy is a major barrier to treatment success, precluding many unfit and elderly patients. Hypomethylating agents (HMA; azacytidine, decitabine) provide a less toxic alternative and have improved treatment options for the unfit. However, only 50% of patients respond to HMA therapy and most eventually acquire resistance, resulting in relapse. Approaches that reduce the toxicity of induction therapy and improve the efficacy of HMA treatment are emerging, e.g. combinations with Venetoclax. Nevertheless, novel combination strategies that improve outcomes in certain mutation subtypes need to be explored. Bisantrene, an anthracene derivative developed over 4 decades ago as a less cardiotoxic chemotherapy alternative to anthracyclines, shows broad activity across multiple solid and haematological malignancies. Several clinical studies in the 1980s showed bisantrene is an effective AML salvage therapy, producing response rates up to 50% without accompanying cardiotoxicity (Rothman J., 2017 Int J Cancer Res Ther;2(2):1-10) In a recent investigator-led Phase II trial of heavily pre-treated relapsed/refractory AML patients, 1/10 achieved a complete remission and 3/10 patients achieved partial remission. Interestingly, all responders were characterised by extramedullary disease (Cannani et al., 2020 Eur J Haematol 2021;106:260-266). We postulate that bisantrene may represent a suitable candidate for combination with HMA in patients unfit for standard induction chemotherapy. To explore this idea, we evaluated the in vitro and in vivo effects of bisantrene both alone and in combination with decitabine. The in vitro activity of bisantrene was initially assessed in a range of human and mouse AML cell lines covering the major molecular and clinical subtypes. All cells were sensitive to bisantrene, showing IC 50 values in the range 16-563 nM. FLT3-ITD + cells in particular were sensitive, while NRAS, KRAS and KIT mutant lines were also responsive. When tested against primary patient AML samples, 15/49 (30.6%) responded strongly to bisantrene treatment ex vivo over the dose range 0.1-1 µM. Bisantrene was shown to induce G1 accumulation and dose-dependent apoptosis in the FLT3-ITD + cell line, MOLM13. When tested in combination with decitabine, pre-treatment with decitabine for 24 h followed by decitabine plus bisantrene for 3 days produced synergistic cytotoxicity in MOLM13, MV4-11 and OCI-AML3 cells. Bisantrene and decitabine together resulted in S phase accumulation in MOLM13 cells and synergistic induction of apoptosis. The in vivo efficacy of bisantrene alone and in combination with decitabine were examined using the MOLM13- luc cell line and a patient derived xenograft (PDX; AML16) (FLT3-ITD +, NPM1, IDH2 and WT1 mutant) in NSG mice. Bisantrene (10 mg/kg i.v. twice/week) significantly reduced leukemic burden and increased median survival compared to vehicle control in MOLM-13- luc engrafted mice, and significantly reduced leukemic burden and increased median survival in a dose and schedule-dependent manner (2.5mg/kg and 5mg/kg, twice or three times weekly) in the PDX model. The combination of bisantrene (5 or 10 mg/kg twice weekly) and decitabine (0.2 mg/kg i.p. 5 days/week) significantly improved survival in MOLM13- luc (Figure 1) and PDX AML16 (Figure 2) engrafted mice, respectively, relative to vehicle control and each single agent. The combination also reduced leukemic infiltration to extramedullary sites (spleen, liver, uterus, brain). These data support that addition of bisantrene to HMA backbone therapy may provide an important new treatment strategy in AML. A prospective Phase Ib/II trial is being planned to evaluate this new therapeutic approach.

Downregulation of RCN1 promotes pyroptosis in acute myeloid leukemia cells.

Reticulocalbin-1 (RCN1) is expressed aberrantly and at a high level in various tumors, including acute myeloid leukemia (AML), yet its impact on AML remains unclear. In this study, we demonstrate that RCN1 knockdown significantly suppresses the viability of bone marrow mononuclear cells (BMMNCs) from AML patients but does not affect the viability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs) from healthy donors in vitro. Downregulation of RCN1 also reduces the viability of AML cell lines. Further studies showed that the RCN1 knockdown upregulates type I interferon (IFN-1) expression and promotes AML cell pyroptosis through caspase-1 and gasdermin D (GSDMD) signaling. Deletion of the mouse Rcn1 gene inhibits the viability of mouse AML cell lines but not the hematopoiesis of mouse bone marrow. In addition, RCN1 downregulation in human AML cells significantly inhibited tumor growth in the NSG mouse xenograft model. Taken together, our results suggest that RCN1 may be a potential target for AML therapy.

KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition.

KMT2C and KMT2D are the most frequently mutated epigenetic genes in human cancers. While KMT2C is identified as a tumor suppressor in acute myeloid leukemia (AML), the role of KMT2D remains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported that KMT2D is downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells with Kmt2d loss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found that KMT2D deficiency leads to the activation of the mTOR pathway in both mouse and human AML cells. Kmt2d directly regulates the expression of Ddit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX-5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML with Kmt2d loss in vivo and extends the survival of leukemic mice. These studies validate KMT2D as a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition.

Abstract A18: STC-15, a novel METTL3 inhibitor, and its combination with Venetoclax confer anti-tumour activity in AML models

Abstract N6-methyladenosine (m6A) is one of the most abundant RNA modifications, which influences mRNA and lncRNA localization, half-life, translation, and splicing. The majority of m6A modifications on cellular mRNAs are deposited by the RNA methyltransferase METTL3. To date, METTL3 has been implicated in the initiation and progression of multiple cancer types, with the highest expression of METTL3 mRNA observed in acute myeloid leukemia (AML). Currently, one line of standard of care therapy for AML patients is Venetoclax, which targets the anti-apoptotic protein BCL2. It was shown that m6A, deposited by METTL3 on BCL2 transcript, affects BCL2 mRNA stability and translation. Storm Therapeutics has developed potent and selective METTL3 inhibitors, including the clinical candidate STC-15. Here, we explore pharmacological inhibition of METTL3 as monotherapy or in combination with Venetoclax in AML models in vitro and in vivo. Sulforhodamine B and CellTiterGloTM assays were used to assess the viability of AML cell lines and patient-derived xenografts (PDXs), respectively, following METTL3 inhibition in vitro. BCL2 protein level was evaluated by Western blotting. SynergyFinder software was used to assess the degree of synergy between METTL3 inhibitors and Venetoclax. Intra-tibial implantation of human-derived AML cells (AML-PDXs) in NSG mice was used to determine single agent and combination therapy efficacy. Multiple AML cells lines and AML-PDXs were sensitive to pharmacological inhibition of METTL3 in vitro, as assessed by loss of viability. Treatment with METTL3 inhibitors led to downregulation of BCL2 protein level in several AML cell lines, as previously suggested by literature. Based on these results, the synergy between METTL3 inhibition and Venetoclax was assessed. Matrix-combination experiments have shown a high degree of synergy between the two drugs (defined by a synergy score >10) in THP-1 and MOLM-13 cell lines. To test METTL3 inhibition as a monotherapy and in combination with Venetoclax in vivo, three AML-PDX studies were initiated. Significantly lower spleen weight was observed in all animals treated with STC-15 or STC-15 + Venetoclax, and reduced number of circulating hCD45+ cells was observed in 2 out of the 3 models. In one of the models, STC-15 monotherapy outperformed Venetoclax (median survival 68 days vs 58 days, respectively), while the combination therapy extended median group survival to 85 days in comparison to 51.5 days in the vehicle group. In conclusion, we demonstrated that METTL3 inhibition results in anti-tumour effects across different AML models. Moreover, we demonstrated a synergistic effect between the novel METTL3 inhibitor STC-15 and Venetoclax, both in vitro and in vivo. These studies provide evidence for the utility of METTL3 inhibitor as a new therapeutic agent to treat AML. Currently, STC-15 is under clinical development (NCT05584111). Citation Format: Lina Vasiliauskaite, Yaara Ofir-Rosenfeld, Mark Albertella, Coralie Hoareau-Aveilla, Jerry McMahon, Oliver Rausch. STC-15, a novel METTL3 inhibitor, and its combination with Venetoclax confer anti-tumour activity in AML models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A18.

Extracellular DEK Treatment Increases Mitochondrial Dysfunction in the Mouse AML Cell line MLL-AF9


 
 
 Acute myeloid leukemia (AML) is the most common kind of acute leukemia and the second most common type of leukemia in adults. Poor outcomes resulting from AML are thought to occur due to the inability to target the small pool of cancer-initiating cells that develop from cells within hematopoietic stem (HSC) and progenitor (HPC) cell compartments in bone marrow. DEK, a nuclear protein that can be secreted under stress conditions, plays a role in regulating HSC and HPC function. Extracellular DEK has been found to improve functional HSC expansion in in-vivo and ex-vivo mouse studies. Moreover, RNAseq experiments suggest that recombinant human DEK treatment causes the upregulation of the antioxidant gene programs. Indeed, DEK treatment reduces total reactive oxygen species (ROS) in human umbilical cord blood HSCs and HPCs. Thus, extracellular DEK enhances normal HSC function through antioxidant programs, but the role of the extracellular DEK in AML is unclear. We hypothesized that recombinant mouse (rm)DEK treatment of the mouse-derived AML cell line MLL-AF9 would affect mitochondrial function since mitochondria are an important source of ROS production. Since ROS production can contribute to mitochondrial dysfunction by causing damage to the organelles, we investigated the effects of DEK signaling on mitochondrial metabolism in MLL-AF9 cells using the Seahorse XF instrument, which can measure changes in metabolic flux. Compared to vehicle-treated control, cells treated with DEK demonstrated a decrease in basal and maximal mitochondrial respiration, proton leak, and non-mitochondrial oxygen consumption. Experiments to explore the effects of DEK treatment on the glycolytic function of MLL-AF9 cells are ongoing. Our data shows DEK treatment of MLL-AF9 cells alters mitochondrial function. In the future, we wish to investigate DEK’s effect on proliferation and colony formation.
 
 

The XPO7/Npat Axis Is a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate below 30%. While AML is a genetically heterogeneous disease, TP53 mutations are among the most powerful risk factors in AML, which underscores the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell, 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss is vulnerable to TP53 deficiency. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture, while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, CRISPR/Cas9-mediated Xpo7 depletion and shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor. In stark contrast, the same sgRNAs which target coding exons including the C-terminal importin-β domain were mostly depleted in Trp53-KO AMLs. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus and mRNA levels of Trp53-targeting genes were significantly decreased upon Xpo7 depletion. Furthermore, Xpo7 depletion resulted in resistance to Selinexor, a XPO1 inhibitor that reportedly blocks Trp53 protein export from the nucleus. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Next, to elucidate the mechanisms underlying the toxic effects observed in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose cytoplasmic/nuclear localization was potentially regulated by Xpo7 in Trp53-KO AML cells. We first performed IP-mass spectrometry using an anti-Xpo7 antibody to identify Xpo7 interacting proteins. Then, we obtained both cytoplasmic and nuclear protein extracts from both Xpo7-WT and -KO AML cells and identified proteins whose localization was significantly altered upon Xpo7 deletion by mass spectrometry. We found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and localized more in the cytoplasm upon Xpo7 depletion in Trp53-KO AMLs to a significant extent. Strikingly, Npat protein levels were robustly upregulated following Trp53 depletion, and Npat depletion significantly delayed the progression of the G1/S transition and proliferation in Trp53-KO AML cells. Finally, to explore the functional significance of the XPO7/NPAT axis in human TP53-mutated AML cells, we assessed the correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM, 2013). Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML, such as AEL.

Polymeric CXCR4 Inhibitors Exhibit Novel Anti-Leukemic Effects

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are common leukemias with a mortality rate estimated to be greater than 50%. This dismal outcome is in part due to current therapeutics. Intensive remission therapy is difficult for patients to tolerate and despite good complete remission rates, is not curative for all patients. In relapse/refractory status, there are even fewer effective therapeutics. There is a strong need for more effective, less toxic therapeutics. The surface receptor CXCR4 has become a recent target of interest, as it is highly expressed on leukemia cells and, via its canonical ligand CXCL12, enables cells to evade chemotherapeutics by localizing them to the protective bone marrow niche. The small molecule inhibitor AMD3100 (plerixafor) disrupts this localization by blocking the CXCL12:CXCR4 interaction and is being investigated for its ability to mobilize leukemia cells to the peripheral blood. Thus, AMD3100 acts as a chemosensitizer, but by itself, AMD3100 does not have anti-leukemic effects. It has been recently shown that CXCR4 has additional non-canonical activities outside of CXCL12 that are vital for leukemia survival and maintenance. Genetic knock out of Cxcr4 in an AML model decreases survival and increases differentiation in a CXCL12 independent manner. This implies that CXCR4 has previously unrecognized activities in leukemia and that inhibitors capable of blocking both CXCR4's canonical and non-canonical activities would more effective therapeutics. We developed a class of polymeric CXCR4 inhibitors (PAMDs) via Michael-type addition of AMD3100 and focused on one analog PAMD-Ch17, which contains additional cholesterol modifications. PAMD-Ch17 was originally designed to delivery siRNAs, but we surprisingly found it had anti-leukemic effects on its own. PAMD-Ch17, but not AMD3100, induces cytotoxicity and promotes differentiation of mouse AML cells in vitro. In colony assays, treatment with PAMD-Ch17 causes a statistically significant decrease in leukemia colony growth while treatment with AMD3100 was indistinguishable from the control. Across a panel of seven human AML and ALL cell lines, PAMD-Ch17 inhibited metabolic activity, a proxy for viability, at the nanomolar doses whereas AMD3100 had no effect even at the highest micromolar dose. As we predict that PAMD-Ch17 is acting through CXCR4, we compared cell surface expression of the receptor to PAMD-Ch17 sensitivity for each cell line. We found that PAMD-Ch17 IC50 was moderately negatively correlated with CXCR4 expression (r= -0.69), suggesting PAMD-CH17 mediates at least some of its effects through CXCR4. To further address the mechanisms of PAMD-Ch17's activity, we cultured Jurkat T-ALL cells with AMD3100 or PAMD-Ch17 at the same dose for 24 hours and stained using the 12G5 CXCR4 monoclonal antibody, which is known to compete with AMD3100 and CXC12. We found that AMD3100 caused a statistically significant decrease in 12G5 staining as compared to control treated cells, but PAMD-Ch17 induced a complete loss of 12G5 staining that was statistically significant as compared to both control and AMD3100 treated cells. PAMD-Ch17, but not AMD3100, also caused a moderate, but significantly decrease in staining with another antibody against the N-terminus of CXCR4, a region not predicted to be affected by AMD3100 or CXCL12. These results indicate that PAMD-Ch17 more effectively blocks the epitope recognized by the 12G5 antibody but may also interact other domains of CXCR4 or decrease its cell surface expression. We also developed a line of PAMD-CH17 resistant Jurkat cells. The resistant cells have an IC50 of at least 2-fold greater than naïve Jurkat cells. Interestingly, the resistant cells are not recognized by the 12G5 monoclonal antibody but are stained by the N-terminus antibody. These results imply that the CXCR4 expressed on resistant cells has an altered conformation that may less effectively bind PAMD-Ch17. Collectively, these data suggest that PAMD-Ch17 more effectively inhibits CXCR4 leading to its novel anti-leukemic effects and support the development of PAMDs for the treatment of leukemia.

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

A Targetable Bone Marrow-Niche Axis for the Treatment of Acute Myeloid Leukemia

Converging lines of evidence show that the endosteal niche within the bone marrow (BM) microenvironment plays a crucial role in the pathogenesis and chemoresistance of myeloid malignancies. Dysplastic cells can exploit niche-dependent, cell non-autonomous pathways to favor their growth. Molecular delineation and targeting of those pathways may help overcome resistance to targeted therapies. In particular, acute myeloid leukemia (AML) remains recalcitrant to conventional chemo- and targeted-therapies leading to relapse and low overall survival rates (5-year survival rate <30%). These shortcomings highlight the urgency of proposing novel and adapted therapies for the disease.Leveraging genetic mouse models, patient-derived xenografts (PDX), and patient samples, herein we show the mechanism and therapeutic targeting of a cell non-autonomous progression pathway in AML. Specifically, we show that conditional ablation of serotonin receptor 1b (HTR1B) in osteoblasts -during or after AML engraftment- hampers or even prevents AML progression, increasing overall survival. Mass spectrometry-based untargeted metabolomics identified the tryptophan catabolite kynurenine (Kyn) as an oncometabolite secreted by AML cells. Remarkably, the AML proliferative pathway that exploits peripheral serotonin signaling in osteoblasts, is not driven by serotonin but by Kyn, which acts as a previously unrecognized HTR1B ligand.To explore in vivo the significance of Kyn for leukemia progression, we inhibited its synthesis by suppressing indoleamine 2,3-dioxygenase-1 (IDO1) activity in mouse and human AML cells. CRISPR-Cas9-mediated Ido1 targeting suppressed AML growth in a dose-dependent manner. Next, we sought to identify the downstream molecular targets of Kyn in human osteoblasts that render the BM niche permissive to AML engraftment and support proliferation of leukemia cells. RNAseq analysis of co-cultures between primary human osteoblasts and leukemia cells shows that AML cells induce a pro-inflammatory remodeling of the osteoblastic niche that depends on HTR1B engagement by Kyn. Among the plethora of secreted pro-inflammatory molecules induced in osteoblasts, AML-secreted Kyn (but not the canonical HTR1B ligand serotonin) induces secretion of the acute-phase protein serum amyloid-A (SAA), which in turn acts in a positive feedback-loop on leukemia cells by increasing IDO1 expression, the rate-limiting enzyme for Kyn synthesis. Acting in an autocrine manner -in a mode distinct from its established immunomodulatory properties in cancer- Kyn activates the aryl hydrocarbon receptor (AHR), thereby supporting further AML proliferation.Highlighting a clinical relevance of these findings, the Kyn-HTR1B-SAA-IDO1 AML-promoting axis identified in leukemia mouse models, is recapitulated in PDX as well as myelodysplastic syndromes (MDS) and AML patients. Preferential and progressive production of Kyn over serotonin by leukemic cells, as well as increased levels of SAA1 in the BM plasma, occur as the disease pathogenesis proceeds from MDS to AML in patients. Moreover, genetic or pharmacological (Epacadostat) IDO1 inhibition hinders AML progression in PDX models. Of note, inhibition of IDO1 synergizes with chemotherapy to reduce AML burden. Overall, these data suggest that the leukemia-osteoblast crosstalk conferred by the Kyn-HTR1B-SAA-IDO1 axis has potential as a therapeutic target. This niche-dependent, cell non-autonomous AML axis, may impact the management of myeloid malignancies, opening also new opportunities for cancer treatment in conjunction with chemo/immunotherapies. DisclosuresBorot: Vor Biopharma: Consultancy, Patents & Royalties: coinventor on issued and pending patent applications licensed to Vor Biopharm. Ali: Vor Biopharma: Consultancy, Patents & Royalties: coinventor on issued and pending patent applications licensed to Vor Biopharm. Roth: UNC: Patents & Royalties: UNC has licensed a patent with B.L.R. listed as an inventor on biased opioid compounds. . Mukherjee: Vor Biopharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: coinventor on issued and pending patent applications licensed to Vor Biopharma. S.M. has equity ownership and is on the Scientific Advisory Board of Vor Biopharma., Research Funding. Rabadan: AimedBio: Membership on an entity's Board of Directors or advisory committees; Genotwin: Other: Raul Rabadan is founder of Genotwin. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Raza: Celgene Inc: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Kura Oncology: Research Funding; Janssen R&D: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau.

CG '806, a Highly Potent First-in-Class Pan-FLT3/BTK Inhibitor, Targets Multiple Clusters of Kinases Operative in Diverse Subtypes of Acute Myeloid Leukemia

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/ Bruton's Tyrosine Kinase (BTK) multi-kinase inhibitor. This small molecule was designed to inhibit the wild type (WT) and C481S mutant forms of BTK, as well as the WT and mutant forms (including the internal tandem duplication, ITD) of the FLT3 receptor tyrosine kinase. Previously (2017 AACR Hematologic Malignancies abstract # 25 and #44), CG'806 was shown to have nM potency against FLT3 mutation-driven (including ITD and point mutations in tyrosine kinase domain) human acute myeloid leukemia (AML) primary patient cells, and in cultured human (eg. MV4-11 and MOLM-13) and mouse (Ba/F3) AML cell lines. CG'806 inhibited FLT3 signaling, induced apoptosis, and eradicated MV4-11 tumors with no observed toxicity in murine xenograft models. Because AML cells demonstrate extreme heterogeneity in gene expression and because FLT3 mutations account for no more than 30% of AML patients, the effect of CG'806 was further evaluated in AML cells without FLT3 mutations. Immunoblotting demonstrated that CG'806 potently decreased phospho-BTK, phospho-aurora kinases A/B/C and phospho-H3S10 without affecting FLT3 activity in AML cells having low/normal expression levels of homozygous WT FLT3 (eg. KG-1 and NOMO-1). Measurement of BrdU incorporation by flow cytometry demonstrated that CG'806 induced G2/M cell cycle arrest or polyploidy and subsequent apoptosis in FLT3 WT AML cell lines. Interestingly, CG'806 was found to induce G0/G1 cell cycle arrest but not polyploidy in FLT3-ITD driven MV4-11 and MOLM-13 cells with no effect on activity of BTK or aurora kinases at concentrations that completely inhibited FLT3 signaling (FLT3/STAT5/ERK). These findings indicate that the molecular mechanism of cell killing by CG'806 is context-dependent and varies in different AML subtypes. Evaluation of enzyme inhibitory activity (IC50) against 583 kinase enzymes, as well as binding affinities (Kd) with 483 kinase proteins, revealed pM to low nM IC50 and Kd values for CG'806 against the FLT3, BTK, AURK, c-Src, c-Met, c-Ret, DDR, TRK and STE/MAPK families of kinases. The ability of CG'806 to target multiple families or clusters of kinases is responsible for its broad efficacy in different AML subtypes driven by different genomic aberrations. Together, these findings illustrate the ability of CG'806 to inhibit entire FLT3 family of kinases, BTK and the AURK families and pathways that are operative in AML, thereby supporting the development of CG'806 for treatment of patients with FLT3-ITD and other subpopulations of patients with AML. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.