Leukemic Growth Research Articles

Gilteritinib Inhibits Acute Myeloid Leukemia Growth via Reduction in Glutamine Uptake and Utilization

The tyrosine kinase inhibitor (TKI) gilteritinib has recently been granted FDA approval for the treatment of relapse/refractory FLT3‐ITD+ AML, yet its mechanism of action is not entirely known. Two recent studies investigating metabolic adaptations during treatment with the FLT3 TKI quizartinib in AML have demonstrated reduced glycolysis or impaired glutaminolysis. Although these studies are conflicting, it suggests that TKIs may influence metabolic changes thereby impacting proliferation and cell viability. Many highly proliferative cells require glutamine as a precursor since they depend on the glutaminolysis pathway for generating building blocks and energy for anabolic processes. Here, we hypothesize that gilteritinib reduces cell viability in FLT3‐ITD+ AML by altering glutamine uptake and metabolism.In our preliminary studies, mice with FLT3‐ITD+ AML (MOLM13 xenograft) were treated with vehicle or gilteritinib (30 mg/kg once daily, five days/week) until leukemic progression. Using an unbiased transcriptomic approach (RNA‐seq) of the leukemic bone marrow, we found that the glutamine transporters (SLC38A1, SLC1A5 and SLC7A5) were significantly downregulated in the gilteritinib‐treated cohort compared to the vehicle cohort (fold change 1.96–4.70, P < 0.0001). Furthermore, expression of metabolic genes was altered in glutamine/aspartate metabolism, glycolysis, and the TCA cycle. Using a resistant xenograft model in which the FLT3‐ITD harbors a mutation in the tyrosine kinase domain at residue D835Y (MOLM13‐RES), SLC38A1 and SLC7A5 were also significantly downregulated after gilteritinib treatment (fold change 0.70–1.28, P < 0.0005), as well as having alterations in the gene expression profile of genes in the aforementioned pathways, although to a lesser extent than MOLM13. In vitro, depleting the cell culture medium of glutamine decreases the cell viability by ~50% in MOLM13 and another FLT3‐ITD+ AML cell line (MV4‐11), indicating that these leukemic cells are addicted to glutamine metabolism as an energy and precursor source. Acute exposure of MOLM13 cells to 100 nM FLT3 TKIs gilteritinib, quizartinib and sorafenib all decrease the transcript of glutamine transporters, with the greatest response from gilteritinib as measured via RT‐PCR (P < 0.01 for all three transporters). In cellular uptake assays, the uptake of glutamine by MOLM13 cells was decreased stepwise to nearly 50% during an 8 h timecourse of 100 nM gilteritinib treatment. This was confirmed using unbiased isotope labeling and tracing in vitro in MOLM13 and MOLM13‐RES cells, which showed reduced glutamine uptake and utilization through the TCA cycle after gilteritinib treatment (15 nM, 24 h). Metabolomics of MOLM13 in vivo samples also revealed alterations in the metabolic pathways. Thus, our data support the premise that gilteritinib downregulates glutamine transporters and alters glutamine metabolism to inhibit cell viability of FLT3‐ITD+ AML.Support or Funding InformationNational Cancer Institute under award R01 CA138744‐08 and the Ohio State University Comprehensive Cancer Center Pelotonia foundation.

DOT1L inhibition is lethal for multiple myeloma due to perturbation of the endoplasmic reticulum stress pathway.

The histone 3 lysine 79 (H3K79) methyltransferase (HMT) DOT1L is known to play a critical role for growth and survival of MLL-rearranged leukemia. Serendipitous observations during high-throughput drug screens indicated that the use of DOT1L inhibitors might be expandable to multiple myeloma (MM). Through pharmacologic and genetic experiments, we could validate that DOT1L is essential for growth and viability of a subset of MM cell lines, in line with a recent report from another team. In vivo activity against established MM xenografts was observed with a novel DOT1L inhibitor. In order to understand the molecular mechanism of the dependency in MM, we examined gene expression changes upon DOT1L inhibition in sensitive and insensitive cell lines and discovered that genes belonging to the endoplasmic reticulum (ER) stress pathway and protein synthesis machinery were specifically suppressed in sensitive cells. Whole-genome CRISPR screens in the presence or absence of a DOT1L inhibitor revealed that concomitant targeting of the H3K4me3 methyltransferase SETD1B increases the effect of DOT1L inhibition. Our results provide a strong basis for further investigating DOT1L and SETD1B as targets in MM.

Tris DBA Ameliorates Accelerated and Severe Lupus Nephritis in Mice by Activating Regulatory T Cells and Autophagy and Inhibiting the NLRP3 Inflammasome.

Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small-molecule palladium complex, has been shown to inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukemia, and multiple myeloma. In the current study, we examined the therapeutic effects of Tris DBA on glomerular cell proliferation, renal inflammation, and immune cells. Treatment of accelerated and severe lupus nephritis (ASLN) mice with Tris DBA resulted in improved renal function, albuminuria, and pathology, including measurements of glomerular cell proliferation, cellular crescents, neutrophils, fibrinoid necrosis, and tubulointerstitial inflammation in the kidneys as well as scoring for glomerulonephritis activity. The treated ASLN mice also showed significantly decreased glomerular IgG, IgM, and C3 deposits. Furthermore, the compound was able to 1) inhibit bone marrow-derived dendritic cell-mediated T cell functions and reduce serum anti-dsDNA autoantibody levels; 2) differentially regulate autophagy and both the priming and activation signals of the NLRP3 inflammasome; and 3) suppress the phosphorylation of JNK, ERK, and p38 MAPK signaling pathways. Tris DBA improved ASLN in mice through immunoregulation by blunting the MAPK (ERK, JNK)-mediated priming signal of the NLRP3 inflammasome and by regulating the autophagy/NLRP3 inflammasome axis. These results suggest that the pure compound may be a drug candidate for treating the accelerated and deteriorated type of lupus nephritis.

Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia

Internal tandem duplication of Fms‐like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD‐transgenic zebrafish, Flt3/ITD knock‐in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient‐derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

Reactive Oxygen Species Drive Proliferation in Acute Myeloid Leukemia via the Glycolytic Regulator PFKFB3.

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a poor clinical outcome. Previously, we showed that overproduction of reactive oxygen species (ROS), arising from constitutive activation of NOX2 oxidase, occurs in >60% of patients with AML and that ROS production promotes proliferation of AML cells. We show here that the process most significantly affected by ROS overproduction is glycolysis. Whole metabolome analysis of 20 human primary AML showed that blasts generating high levels of ROS have increased glucose uptake and correspondingly increased glucose metabolism. In support of this, exogenous ROS increased glucose consumption while inhibition of NOX2 oxidase decreased glucose consumption. Mechanistically, ROS promoted uncoupling protein 2 (UCP2) protein expression and phosphorylation of AMPK, upregulating the expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Overexpression of PFKFB3 promoted glucose uptake and cell proliferation, whereas downregulation of PFKFB3 strongly suppressed leukemia growth both in vitro and in vivo in the NSG model. These experiments provide direct evidence that oxidase-derived ROS promotes the growth of leukemia cells via the glycolytic regulator PFKFB3. Targeting PFKFB3 may therefore present a new mode of therapy for this disease with a poor outcome. SIGNIFICANCE: These findings show that ROS generated by NOX2 in AML cells promotes glycolysis by activating PFKFB3 and suggest PFKFB3 as a novel therapeutic target in AML.

Down-regulating NQO1 promotes cellular proliferation in K562 cells via elevating DNA synthesis

BackgroundNQO1 protein acts as a cellular protective system, on account of its role as a quinone reductase and redox regulator. Nonetheless, new NQO1 roles are emerging—including its regulation of the cellular proliferation of many tumor cells—and this enzyme has been found to relate to the incidence of various diseases, including chronic myeloid leukemia. However, the mechanisms through which NQO1 influences leukemia progression remain unclear. Martial and methodsThe current study looks to name NQO1 as a novel molecular target that modulates DNA synthesis and chronic myeloid leukemia growth. Results and conclusionOur results indicate that the frequency of the T allele of NQO1 polymorphism in chronic myeloid leukemia patients is higher than that among healthy East Asian individuals (0.492 vs. 0.419) and much higher than the average level of the general population (0.492 vs. 0.289) (1000 Genomes). Functionally, NQO1 knockdown increases the protein expression of the TOP2A and MCM complex, and consequently promotes DNA synthesis and K562 cell growth. NQO1 knockdown also promotes tumorigenesis in a xenograft model. NQO1 overexpression, on the other hand, was found to have the opposite effects. SignificanceOur results show that NQO1 downregulation promotes K562 cellular proliferation via the elevation of DNA synthesis.

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.

PBX3 Promotes Tumor Growth and Angiogenesis via Activation of AT1R/VEGFR2 Pathway in Papillary Thyroid Carcinoma

PBX3 (Pre-B-cell leukemia homeobox 3) had been considered to be a multifunctional oncogene which involved in tumor growth, invasion, and metastasis in leukemia and some solid tumors. However, the contribution of PBX3 to papillary thyroid carcinoma (PTC) remains unclear. In this study, we found that PBX3 expression was significantly upregulated in PTC tissues compared to adjacent normal tissues, and high levels of PBX3 were correlated with tumor size, lymphatic metastasis, TMN stage, and poor prognosis of PTC patients. Overexpression of PBX3 in PTC cell lines promoted cell proliferation. Consistently, knockdown of PBX3 by shRNA induced cell cycle arrest at G0/G1 phase, and inhibited angiogenesis and tumor growth in vitro and in vivo. Furthermore, PBX3 promoted PTC cell proliferation and angiogenesis through activation of AT1R/VEGFR2 pathway while overexpression of AT1R and treatment with VEGFA reversed PBX3-shRNA-induced decreased phosphorylation of VEGFR2 and its downstream (ERK1/2, AKT and Src). It demonstrated that PBX3 could be used as a potential prognostic biomarker and therapeutic target for PTC.

1,2,3-Triazole-Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic investigation of apoptosis induction in multiple myeloma RPMI-8226

A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and SR leukemia cell lines by 99.73% and 94.95% at 10 μM, respectively. Also, it inhibited the growth of M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test concentration. 4d showed IC50 values less than 1 μM on six types of tumor cells and high selectivity index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound 4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner. Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.

Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.

Effects of DNA Methylation on Leukemia Cell Proliferation.

In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation. Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1. DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased. These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

The stem cell–specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia

AbstractHOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA– and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)–mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.

Effective targeting of NAMPT in patient-derived xenograft models of high-risk pediatric acute lymphoblastic leukemia.

The prognosis for children diagnosed with high-risk acute lymphoblastic leukemia (ALL) remains suboptimal, and more potent and less toxic treatments are urgently needed. We investigated the efficacy of a novel nicotinamide phosphoribosyltransferase inhibitor, OT-82, against a panel of patient-derived xenografts (PDXs) established from high-risk and poor outcome pediatric ALL cases. OT-82 was well-tolerated and demonstrated impressive single agent in vivo efficacy, achieving significant leukemia growth delay in 95% (20/21) and disease regression in 86% (18/21) of PDXs. In addition, OT-82 enhanced the efficacy of the established drugs cytarabine and dasatinib and, as a single agent, showed similar efficacy as an induction-type regimen combining three drugs used to treat pediatric ALL. OT-82 exerted its antileukemic action by depleting NAD+ and ATP, inhibiting the NAD+-requiring DNA damage repair enzyme PARP-1, increasing mitochondrial ROS levels and inducing DNA damage, culminating in apoptosis induction. OT-82 sensitivity was associated with the occurrence of mutations in major DNA damage response genes, while OT-82 resistance was characterized by high expression levels of CD38. In conclusion, our study provides evidence that OT-82, as a single agent, and in combination with established drugs, is a promising new therapeutic strategy for a broad spectrum of high-risk pediatric ALL for which improved therapies are urgently needed.

FLI1 promotes protein translation via the transcriptional regulation of MKNK1 expression.

The disruption of protein translation machinery is a common feature of cancer initiation and progression, and drugs that target protein translation offer new avenues for therapy. The translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is induced in a number of cancer cell lines and is one such candidate for therapeutic intervention. Friend leukemia integration 1 (FLI1) is a potent oncogenic transcription factor that promotes various types of cancer by promoting several hallmarks of cancer progression. FLI1 has recently been implicated in protein translation through yet unknown mechanisms. This study identified a positive association between FLI1 expression and mitogen-activated protein kinase (MAPK)-interacting serine/threonine kinase1 (MKNK1), the immediate upstream regulator of the eIF4E initiation factor. The short hairpin RNA (shRNA)-mediated silencing or overexpression of FLI1 in leukemic cell lines downregulated or upregulated MKNK1 expression, respectively. Promoter analysis identified a potent FLI1 binding site in the regulatory region of the MKNK1 promoter. In transient transfection experiments, FLI1 increased MKNK1 promoter activity, which was blocked by mutating the FLI1 binding site. FLI1 specifically affected the expression of MKNK1, but not that of MKNK2. The siRNA-mediated downregulation of MKNK1 downregulated the expression of survivin (BIRC5) and significantly suppressed cell proliferation in culture. FLI1 inhibitory compounds were shown to downregulate this oncogene through the suppression of MAPK/extracellular-regulated kinase (ERK) signaling and the subsequent activation of miR-145, leading to a lower MKNK1 expression and the suppression of leukemic growth. These results uncover a critical role for FLI1 in the control of protein translation and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy.

Abstract C12: Lipids signaling contributes to glucose-independent metabolic adaptation in pancreatic ductal adenocarcinoma

Abstract Increasing researches verify that mitochondrial oxidase phosphorylation (OXPHOS) plays remarkable roles in cancer initiation, development, and drug resistance. Our previous study identified that preclinic OXPHOS inhibitor IACS-010759 significantly suppressed cancer cell growth in brain cancer and acute myeloid leukemia (AML) in vivo and in vitro with very low compound concentration. In pancreatic ductal adenocarcinoma (PDAC), there were actually different responses to OXPHOS inhibition in glucose-independent conditions. Using patient-derived xenograft (PDX) model, we identified sensitive and resistant groups to IACS-010759, and there were dramatic metabolic changes to feed-back compound resistance. By metabolomics analysis, we found a novel lipids-signaling activation mechanism that is responsible for the adaptive metabolic resistance. Furthermore, combination of OXPHOS inhibitor and specific lipids pathway inhibitor showed significant cancer cell growth suppression in resistant PDAC types. Our findings uncovered a novel adaptive metabolism in drug resistance and provide new thoughts on therapy strategy in PDAC. Citation Format: Ziheng Chen, Jintan Liu, I-Lin Ho, Johnathan Rose, Liang Yan, Wantong Yao, Andrea Viale, Alessandro Carugo, Haoqiang Ying, Giulio Draetta. Lipids signaling contributes to glucose-independent metabolic adaptation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C12.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

A CRISPR/Cas9-Generated Murine Model Reveals Cooperation between BCR Signaling and CDKN2A/2B and TP53 Disruption in Richter Syndrome

B cell receptor (BCR) signals generated by chronic interactions with external autoantigens or cell-autonomous BCR-BCR interactions play a critical role in the pathogenesis of CLL (Dühren-von Minden M et al, Nature 2012; Iacovelli S et al, Blood 2015). However, the role of these signals in regulating CLL cell responses and in particular in regulating CLL cell proliferation has still not been established. To address this issue, we first reanalyzed a previously generated dataset of human CLL cells stimulated with immobilized anti-IgM (Dal Bo M et al, Oncotarget 2015), which revealed differential expression of a number of genes involved in regulating the G1 phase of the cell cycle. The observed changes included upregulation of positive regulators of the cell cycle, such as CCND1, CCND2 and CDK4, but also upregulation of negative regulators, such as the cell cycle inhibitors CDKN1A, CDKN2A and CDKN2B. Changes in the expression of these genes were further validated by RQ-PCR analysis of a separate set of anti-IgM-stimulated human CLL cells (n=12), as well as by analysis of murine Eμ-TCL1-derived CLL cells stimulated with their cognate antigen phosphatidylcholine (PtC) (n=5). To investigate whether these genes are also induced by cell-autonomous BCR-BCR interactions, we analyzed the same samples following treatment with the SYK inhibitor entospletinib or the BTK inhibitor ibrutinib (1 μM each for 20 hours). A modest reduction in the levels of the cell cycle inhibitor CDKN2B but no change in the levels of any of the positive cell cycle regulators was observed, suggesting that only signals generated by external antigens can induce CLL cells to enter the cell cycle. The finding that the negative regulators CDKN1A, CDKN2A and CDKN2B are induced upon BCR engagement with external ligand appeared particularly interesting, considering that these genes are frequently downregulated in cases with Richter transformation because of co-existing genetic lesions in CDKN2A/2B and TP53, which is a key transcriptional activator of CDKN1A. Moreover, these findings suggested that genetic lesions in CDKN2A, CDKN2B and TP53 could contribute to Richter transformation by increasing the proliferation of leukemic cells stimulated with external autoantigen. To further address this possibility, we simultaneously targeted CDKN2A, CDKN2B and TP53 by CRISPR/Cas9 in primary Eμ-TCL1-derived murine CLL cells expressing an autoreactive BCR with specificity for the PtC autoantigen. The Cas9-edited and non-edited CLL cells were then expanded in immunodeficient NSG mice and were subsequently transferred in syngeneic recipients (n=6-8 per group) to evaluate the effects of CDKN2A/2B/TP53 knockdown on leukemia growth and proliferation. Combined targeting of CDKN2A, CDKN2B and TP53 resulted in accelerated leukemia growth, as evidenced by the significantly higher number of leukemic cells in the peritoneal cavity, peripheral blood and spleen of mice that received Cas9-edited vs wild type CLL cells (Figure 1A). The Cas9-edited leukemic cells showed markedly reduced expression of CDKN1A and CDKN2B protein (CDKN2A expression was not assessed in these experiments) and significantly higher expression of the proliferative markers Ki67, BrdU and phospho-histone H3 compared to non-edited leukemic cells. Moreover, indel analysis by amplicon capillary electrophoresis showed an increase in the ratio of CDKN2B mutant and TP53 mutant alleles vs wild type alleles during in vivo propagation, further suggesting that cells with combined CDKN2A/2B/TP53 deficiency have a growth advantage over wild type CLL cells (Figure 1B). A separate experiment with independent knockdown of CDKN2A/2B and TP53 also revealed selection of the mutant alleles during in vivo propagation, although only CDKN2A/2B knockdown resulted in a significant increase in tumor burden (Figure 1C and 1D). In conclusion, these data demonstrate that BCR interactions with external autoantigens induce both positive and negative regulators of the cell cycle and suggest that genetic lesions associated with Richter transformation cooperate with BCR signals by downregulating the negative regulators and facilitating cell cycle progression. Disclosures No relevant conflicts of interest to declare.

Endogenous Retinoid X Receptor Ligands Act As Tumor Suppressors in MLL-AF9 Mouse Leukemia

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are ligand-activated transcription factors that influence hematopoietic stem cell self-renewal and differentiation. In normal hematopoiesis, RARA and RXRA are dynamically regulated during myeloid maturation, with highest expression in mature neutrophils. In acute myeloid leukemia (AML) RARA and RXRA have parallel expression among AML subtypes, with the highest expression in M4/M5 myelomonocytic and monocytic subtypes. Using a murine reporter assay, we found that natural RXRA ligands, but not RARA ligands, were present in vivo in primary mouse myelomonocytic leukemia cells (derived with an MLL-AF9 retrovirus) and acted as tumor suppressors. RXR ligands were absent in erythroleukemia (derived with TLS-ERG) or T-cell leukemia (derived with activated Notch1), suggesting the presence of natural RXRA ligands specifically in myelomonocytic leukemia. Moreover, we found that deletion of Rxra and Rxrb was naturally selected in MLL-AF9, but not in Notch1 or TLS-ERG leukemias, and that loss of Rxra and Rxrb accelerated leukemic growth. This suggests that Rxrs act as tumor suppressors, but only when exposed to natural ligands. In MLL-AF9 derived leukemia cells, pharmacologic treatment with single-agent retinoid led to modest growth inhibition due to non-permissive activity of the RARA:RXR heterodimer, whereas concurrent activation of both RARA, with all-trans retinoic acid (a pan-RAR ligand), and RXR, by bexarotene (a pan-RXR ligand), enabled efficient co-repressor release and synergistic leukemic apoptosis. We observed that co-repressors release (SMRT/NCoR) from the RARA:RXRA heterodimer was specifically associated with RARA activation, whereas growth inhibition required RXR binding, and this occurred through apoptosis rather than maturation. Generating a series of RXRA mutant proteins we demonstrated the active contribution of RXRA to the activity of the RARA:RXRA heterodimer, specifically requiring the activation domains (AF1 and AF2) and the ability to recruit co-activator to the RXRA element. Finally, we observed a significant dose-dependent effect of combination ATRA and bexarotene treatment in in vivo in MLL-AF9 leukemic mice with a striking reduction in the tumor burden of treated mice compared to the control cohort. These data provide a strategy for clinical retinoid therapies in leukemias beyond acute promyelocytic leukemia and provide a mechanism for integrating retinoid therapy into future clinical trials. Disclosures No relevant conflicts of interest to declare.

The Distinctive Metabolic Environment of the Bone Marrow Niche Drives Leukemia Chemoresistance

Chemoresistance is the difference between remission and cure in cancer patients and is particularly evident in acute myeloid leukemia (AML) where complete remissions are common, but few are cured. Genetic analysis in patients points to residual founder clones persisting through therapy and ultimately causing relapse. However, most of these genetic alterations are currently not amenable to targeted therapies. We hypothesized that residual chemoresistant cells must pass through extreme metabolic challenges when under selection from chemotherapy and surrounded by massive cell death. Our goal was to define the distinctive metabolic profile of AML cells as they pass through that time of extreme selection pressure and determine whether metabolic pathway manipulation would reveal an exploitable vulnerability for therapy. We used in vivo imaging to define the kinetics of leukemic growth, response to chemotherapy and relapse in the mouse. We reasoned that the moment of maximal response immediately following chemotherapy represents a time when the metabolic milieu is maximally hostile and therefore a distinctive selection moment that the cells ultimately causing relapse must pass through. We captured cells at that time and conducted unbiased metabolic profiling, which revealed a metabolite profile that was distinct from AML cells during pre-chemo growth or late post-chemo relapse. Metabolite enrichment analysis revealed hyperactivation of glutamine metabolism during the extreme selection period, with chemoresistant cells showing high levels of glutamine, glutamate and aspartate. Inhibition of glutamine metabolism using the glutamine analog 6-diazo-5-oxo-L-norleucine specifically during the window of maximal selection pressure reduced the number of chemoresistant cells and improved survival in mouse models of AML. Unexpectedly, we found that residual AML cells were not sensitive to inhibition of the enzyme glutaminase, a target currently under clinical investigation for AML. In vivo metabolic tracer analysis instead revealed that chemoresistant AML cells mainly depend on glutamine nitrogen to fuel pyrimidine synthesis. Aspartate, another essential metabolite for pyrimidine synthesis, can be made from glutamine through the Krebs cycle. Interestingly, we detected almost no glutamine carbon entering the Krebs cycle in vivo even though labeled aspartate was found, indicating a different aspartate source. We found that aspartate levels in peripheral blood plasma are very low compared to those in bone marrow plasma, and explored the possibility of a local bone marrow source. Single cell RNA sequencing of bone marrow stromal cells (BMSC) revealed that CXCL12-positive BMSC express high levels of aspartate synthesis genes and transporters, which further increased in the presence of AML cells. In vitro co-cultures showed that BMSC convert glutamine to aspartate, and transfer this to AML cells. Genetic inhibition of aspartate synthesis in BMSC sensitized AML cells to chemotherapy, showing the importance of this metabolic crosstalk. We propose that induction of a timed metabolic collapse targeting AML cells both directly and indirectly through the bone marrow niche can prevent development of chemoresistance and improve treatment outcomes. Figure Disclosures Scadden: Novartis: Other: Sponsored research; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

R-2HG/KDM2B/RIPK1 Signaling Mediates Necroptosis in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders and manifested as ineffective hematopoiesis, refractory cytopenia and a propensity to evolve into acute myeloid leukemia (AML). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in both MDS and AML. Mutated IDH produces R-2-hydroxyglutarate (R-2HG) which inhibits multiple α-ketoglutarate/α-KG-dependent dioxygenases by competing against α-KG binding. Recent studies have demonstrated that R-2-HG can abrogates leukemic growth and induce leukemia cell death. Several nonapoptotic cell death have been identified, including phosphoribosyl pyrophosphate (PPRP)-1 mediated necrosis, pyroptosis, ferroptosis and necroptosis. By now, the specific type of cell death by which R-2-HG exerts anti-tumor effects is still unknown. Results: (1) (2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1, THP-1, Molm-13, HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose- and time-dependent manner. The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines, and the inhibition rate was related to the dose of doxycycline. (2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest. (2) R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells. The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in (2R)-Octyl-2-HG groups, and the expression of RIPK1 gene was increased in all three (SKM-1, NOMO-1 and MA9.3ITD) (2R)-Octyl-2-HG groups. After treatment with (2R)-Octyl-2-HG, the mRNA and protein expression levels of RIPK1 gene were increased. (3) R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis. Within 10 hours after treatment with (2R)-Octyl-2-HG, cell membrane permeability was disrupted, interactions between RIPK1 and caspase 8 increased, as well as phosphorylated MLKL level. However, caspase activity did not increase significantly, suggesting that necroptosis occurs earlier than apoptosis. With RIPK1 inhibitor Necrostatin-1 and (2R)-Octyl-2-HG co-treating cells, proliferation inhibition was reduced, cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form. The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus, suggesting that RIPK1-dependent necroptosis is involved in cell death caused by (2R)-Octyl-2-HG. (4) In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK. In RIPK1 shRNA MDS mice, the tumor burden showed a decreasing trend, but did not show a significant change in the R-2-HG treatment group. Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+ cells in bone marrow. (5) Low RIPK1 expression predicts poor prognosis in MDS patients. Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls. Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival (OS) than patients with higher RIPK1 expression levels. MDS patients with lower RIPK1 expression levels progress to leukemia more frequently. (6) Inhibition of KDM2B induces necroptosis independently. Western blot assay shows the knockdown of KDM2B, upregulation of RIPK1 and increased levels of p-MLKL. Analysis of cell numbers showed that proliferation ceased from 4 days after doxycycline treatment onwards in shKDM2B cells. Co-IP assay shows the formation of RIPK1-caspase8 complex. Conclusion: This study confirmed that R-2-HG inhibited the viability of MDS and AML cells. R-2-HG increased the expression of RIPK1 in MDS cells, inducing necroptosis. Necroptosis occurred earlier than apoptosis. Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells. Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML. MDS patients with low expression of RIPK1 was more likely to progress to leukemia. Inhibition of KDM2B can induce necroptosis independently. Disclosures No relevant conflicts of interest to declare.