Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Targeting MCL-1 dysregulates cell metabolism and leukemia-stroma interactions and resensitizes acute myeloid leukemia to BCL-2 inhibition.

MCL-1 and BCL-2 are both frequently overexpressed in acute myeloid leukemia and critical for the survival of acute myeloid leukemia cells and acute myeloid leukemia stem cells. MCL-1 is a key factor in venetoclax resistance. Using genetic and pharmacological approaches, we discovered that MCL-1 regulates leukemia cell bioenergetics and carbohydrate metabolisms, including the TCA cycle, glycolysis and pentose phosphate pathway and modulates cell adhesion proteins and leukemia-stromal interactions. Inhibition of MCL-1 sensitizes to BCL-2 inhibition in acute myeloid leukemia cells and acute myeloid leukemia stem/progenitor cells, including those with intrinsic and acquired resistance to venetoclax through cooperative release of pro-apoptotic BIM, BAX, and BAK from binding to anti-apoptotic BCL-2 proteins and inhibition of cell metabolism and key stromal microenvironmental mechanisms. The combined inhibition of MCL-1 by MCL-1 inhibitor AZD5991 or CDK9 inhibitor AZD4573 and BCL-2 by venetoclax greatly extended survival of mice bearing patient-derived xenografts established from an acute myeloid leukemia patient who acquired resistance to venetoclax/decitabine. These results demonstrate that co-targeting MCL-1 and BCL-2 improves the efficacy of and overcomes preexisting and acquired resistance to BCL-2 inhibition. Activation of metabolomic pathways and leukemia-stroma interactions are newly discovered functions of MCL-1 in acute myeloid leukemia, which are independent from canonical regulation of apoptosis by MCL-1. Our data provide new mechanisms of synergy and rationale for co-targeting MCL-1 and BCL-2 clinically in patients with acute myeloid leukemia and potentially other cancers.

Combined inhibition of MDM2 and BCR-ABL1 tyrosine kinase targets chronic myeloid leukemia stem/progenitor cells in a murine model.

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45+ and linage-SCA-1+C-KIT+ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1+C-KIT+ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.

Concomitant targeting of BCL2 with venetoclax and MAPK signaling with cobimetinib in acute myeloid leukemia models.

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.

Integrated genomic analysis of recurrence-associated small non-coding RNAs in oesophageal cancer.

OBJECTIVE: Oesophageal squamous cell carcinoma (ESCC) is a heterogeneous disease with variable outcomes that are challenging to predict. A better understanding of the biology of ESCC recurrence is needed to improve patient care. Our goal was to identify small non-coding RNAs (sncRNAs) that could predict the likelihood of recurrence after surgical resection and to uncover potential molecular mechanisms that dictate clinical heterogeneity. DESIGN: We developed a robust prediction model for recurrence based on the analysis of the expression profile data of sncRNAs from 108 fresh frozen ESCC specimens as a discovery set and assessment of the associations between sncRNAs and recurrence-free survival (RFS). We also evaluated the mechanistic and therapeutic implications of sncRNA obtained through integrated analysis from multiple datasets. RESULTS: We developed a risk assessment score (RAS) for recurrence with three sncRNAs (microRNA (miR)-223, miR-1269a and nc886) whose expression was significantly associated with RFS in the discovery cohort (n=108). RAS was validated in an independent cohort of 512 patients. In multivariable analysis, RAS was an independent predictor of recurrence (HR, 2.27; 95% CI, 1.26 to 4.09; p=0.007). This signature implies the expression of ΔNp63 and multiple alterations of driver genes like PIK3CA. We suggested therapeutic potentials of immune checkpoint inhibitors in low-risk patients, and Polo-like kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and histone deacetylase inhibitors in high-risk patients. CONCLUSION: We developed an easy-to-use prognostic model with three sncRNAs as robust prognostic markers for postoperative recurrence of ESCC. We anticipate that such a stratified and systematic, tumour-specific biological approach will potentially contribute to significant improvement in ESCC treatment.

Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.

Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations.

Apoptosis repressor with caspase recruitment domain modulates second mitochondrial-derived activator of caspases mimetic-induced cell death through BIRC2/MAP3K14 signalling in acute myeloid leukaemia.

Overexpression of the apoptosis repressor with caspase recruitment domain (ARC, also termed NOL3) protein predicts adverse outcome in patients with acute myeloid leukaemia (AML) and confers drug resistance to AML cells. The second mitochondrial-derived activator of caspases (SMAC, also termed DIABLO) mimetic, birinapant, promotes extrinsic apoptosis in AML cells. SMAC mimetics induce cleavage of cellular inhibitor of apoptosis (cIAP) proteins, leading to stabilization of the nuclear factor-κB (NF-κB)-inducing kinase (MAP3K14, also termed NIK) and activation of non-canonical NF-κB signalling. To enhance the therapeutic potential of SMAC mimetics in AML, we investigated the regulation and role of ARC in birinapant-induced apoptosis. We showed that birinapant increases ARC in AML and bone marrow-derived mesenchymal stromal cells (MSCs). Downregulation of MAP3K14 by siRNA decreased ARC levels and suppressed birinapant-induced ARC increase. Reverse-phase protein array analysis of 511 samples from newly diagnosed AML patients showed that BIRC2 (also termed cIAP1) and ARC were inversely correlated. Knockdown of ARC sensitized, while overexpression attenuated, birinapant-induced apoptosis. Furthermore, ARC knockdown in MSCs sensitized co-cultured AML cells to birinapant-induced apoptosis. Our data demonstrate that ARC is regulated via BIRC2/MAP3K14 signalling and its overexpression in AML or MSCs can function as a resistant factor to birinapant-induced leukaemia cell death, suggesting that strategies to inhibit ARC will improve the therapeutic potential of SMAC mimetics.

Survivin is highly expressed in CD34(+)38(-) leukemic stem/progenitor cells and predicts poor clinical outcomes in AML.

Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34(+)38(-) AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34(+)38(-) AML stem/progenitor cells than in bulk blasts and total CD34(+) AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Expression of ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein, is strongly prognostic in AML.

Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.

Immune checkpoint B7-H3 is a therapeutic vulnerability in prostate cancer harboring PTEN and TP53 deficiencies.

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML.

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Maximal Activation of Apoptosis Signaling by Cotargeting Antiapoptotic Proteins in BH3 Mimetic-Resistant AML and AML Stem Cells.

MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells.

XIAP antisense oligonucleotide (AEG35156) achieves target knockdown and induces apoptosis preferentially in CD34+38- cells in a phase 1/2 study of patients with relapsed/refractory AML.

XIAP, a potent caspase inhibitor, is highly expressed in acute myeloid leukemia (AML) cells and contributes to chemoresistance. A multi-center phase 1/2 trial of XIAP antisense oligonucleotide AEG35156 in combination with idarubicin/cytarabine was conducted in 56 patients with relapsed/refractory AML. Herein we report the pharmacodynamic studies of the patients enrolled at M. D. Anderson Cancer Center. A total of 13 patients were enrolled in our institution: five in phase 1 (12-350 mg/m² AEG35156) and eight in phase 2 (350 mg/m² AEG35156) of the protocol. AEG35156 was administered on 3 consecutive days and then weekly up to a maximum of 35 days. Blood samples were collected from patients on days 1 through 5 and on day 28-35 post-chemotherapy for detection of XIAP levels and apoptosis. AEG35156 treatment led to dose-dependent decreases of XIAP mRNA levels (42-100% reduction in phase 2 patients). XIAP protein levels were reduced in all five samples measured. Apoptosis induction was detected in 1/4 phase 1 and 4/5 phase 2 patients. Importantly, apoptosis was most pronounced in CD34+38- AML stem cells and all phase 2 patients showing apoptosis induction in CD34+38- cells achieved response. We conclude that at 350 mg/m², AEG35156 is effective in knocking down XIAP in circulating blasts accompanied by the preferential induction of apoptosis in CD34+38- AML stem cells.

Extended live-cell barcoding approach for multiplexed mass cytometry.

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. Sample pooling after prior surface staining termed live-cell barcoding is more desirable than intracellular barcoding, where samples are pooled after fixation and permeabilization, since it does not depend on fixation-sensitive antigenic epitopes. In live-cell barcoding, the general approach uses two tags per sample out of a pool of antibodies paired with five palladium (Pd) isotopes in order to preserve appreciable signal-to-noise ratios and achieve higher yields after sample deconvolution. The number of samples that can be pooled in an experiment using live-cell barcoding is limited, due to weak signal intensities associated with Pd isotopes and the relatively low number of available tags. Here, we describe a novel barcoding technique utilizing 10 different tags, seven cadmium (Cd) tags and three Pd tags, with superior signal intensities that do not impinge on lanthanide detection, which enables enhanced pooling of samples with multiple experimental conditions and markedly enhances sample throughput.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

An ARC-Regulated IL1ß/Cox-2/PGE2/ß-Catenin/ARC Circuit Controls Leukemia-Microenvironment Interactions and Confers Drug Resistance in AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is a strong independent adverse prognostic marker in acute myeloid leukemia (AML). We previously reported that ARC regulates leukemia-microenvironment interactions through the NFκB/IL1ß signaling network. Malignant cells have been reported to release IL1ß, which induces PGE2 synthesis in mesenchymal stromal cells (MSC), in turn activating ß-catenin signaling and inducing the cancer stem cell phenotype. Although Cox-2 and its enzymatic product PGE2 play major roles in inflammation and cancer, the regulation and role of PGE2 in AML are largely unknown. Here, we report that AML-MSC cocultures greatly increase Cox-2 expression in MSC and PGE2 production in an ARC/IL1ß-dependent manner. PGE2 induced the expression of ß-catenin, which regulated ARC and augmented chemoresistance in AML cells; inhibition of ß-catenin decreased ARC and sensitized AML cells to chemotherapy. NOD/SCIDIL2RγNull-3/GM/SF mice transplanted with ARC-knockdown AML cells had significantly lower leukemia burden, lower serum levels of IL1ß/PGE2, and lower tissue human ARC and ß-catenin levels, prolonged survival, and increased sensitivity to chemotherapy than controls. Collectively, we present a new mechanism of action of antiapoptotic ARC by which ARC regulates PGE2 production in the tumor microenvironment and microenvironment-mediated chemoresistance in AML.Significance: The antiapoptotic protein ARC promotes AML aggressiveness by enabling detrimental cross-talk with bone marrow mesenchymal stromal cells.

Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia.

In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.