Genomic landscape of Down syndrome-associated acute lymphoblastic leukemia.

Trisomy 21, the genetic cause of Down syndrome (DS), is the most common congenital chromosomal anomaly. It is associated with a 20-fold increased risk of acute lymphoblastic leukemia (ALL) during childhood and results in distinctive leukemia biology. To comprehensively define the genomic landscape of DS-ALL, we performed whole-genome sequencing and whole-transcriptome sequencing (RNA-Seq) on 295 cases. Our integrated genomic analyses identified 15 molecular subtypes of DS-ALL, with marked enrichment of CRLF2-r, IGH::IGF2BP1, and C/EBP altered (C/EBPalt) subtypes compared with 2257 non-DS-ALL cases. We observed abnormal activation of the CEBPD, CEBPA, and CEBPE genes in 10.5% of DS-ALL cases via a variety of genomic mechanisms, including chromosomal rearrangements and noncoding mutations leading to enhancer hijacking. A total of 42.3% of C/EBP-activated DS-ALL also have concomitant FLT3 point mutations or insertions/deletions, compared with 4.1% in other subtypes. CEBPD overexpression enhanced the differentiation of mouse hematopoietic progenitor cells into pro-B cells in vitro, particularly in a DS genetic background. Notably, recombination-activating gene-mediated somatic genomic abnormalities were common in DS-ALL, accounting for a median of 27.5% of structural alterations, compared with 7.7% in non-DS-ALL. Unsupervised hierarchical clustering analyses of CRLF2-rearranged DS-ALL identified substantial heterogeneity within this group, with the BCR::ABL1-like subset linked to an inferior event-free survival, even after adjusting for known clinical risk factors. These results provide important insights into the biology of DS-ALL and point to opportunities for targeted therapy and treatment individualization.

CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome.

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.

Defining the transcriptional control of pediatric AML highlights RARA as a superenhancer-regulated druggable dependency.

Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)-rearranged samples were also distinct from non-KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE- samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE- patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

Inhibition of the MAP2K7-JNK pathway with 5Z-7-oxozeaenol induces apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients.

Enhancer polymorphisms at the IKZF1 susceptibility locus for acute lymphoblastic leukemia impact B-cell proliferation and differentiation in both Down syndrome and non-Down syndrome genetic backgrounds.

Children with Down syndrome have an approximately 10-fold increased risk of developing acute lymphoblastic leukemia and this risk is influenced by inherited genetic variation. Genome-wide association studies have identified IKZF1 as a strong acute lymphoblastic leukemia susceptibility locus in children both with and without Down syndrome, with association signals reported at rs4132601 in non-Down syndrome and rs58923657 in individuals with Down syndrome (r2 = 0.98 for these two loci). Expression quantitative trait locus analysis in non-Down syndrome lymphoblastoid cell lines has demonstrated an association between the rs4132601 risk allele and decreased IKZF1 mRNA levels. In this study, we provide further mechanistic evidence linking the region encompassing IKZF1-associated polymorphisms to pro-leukemogenic effects in both human lymphoblastoid cell lines and murine hematopoietic stem cells. CRISPR/Cas9-mediated deletion of the region encompassing the rs17133807 major allele (r2 with rs58923657 = 0.97) resulted in significant reduction of IKZF1 mRNA levels in lymphoblastoid cell lines, with a greater effect in Down syndrome versus non-Down syndrome cells. Since rs17133807 is highly conserved in mammals, we also evaluated the orthologous murine locus at rs263378223, in hematopoietic stem cells from the Dp16(1)Yey mouse model of Down syndrome as well as non-Down syndrome control mice. Homozygous deletion of the region encompassing rs263378223 resulted in significantly reduced Ikzf1 mRNA, confirming that this polymorphism maps to a strong murine Ikzf1 enhancer, and resulted in increased B-lymphoid colony growth and decreased B-lineage differentiation. Our results suggest that both the region encompassing rs17133807 and its conserved orthologous mouse locus have functional effects that may mediate increased leukemia susceptibility in both the Down syndrome and non-Down syndrome genetic backgrounds.

Metabolomic profiling identifies pathways associated with minimal residual disease in childhood acute lymphoblastic leukaemia.

BACKGROUND: End-induction minimal residual disease (MRD) is the strongest predictor of relapse in paediatric acute lymphoblastic leukaemia (ALL), but an understanding of the biological pathways underlying early treatment response remains elusive. We hypothesized that metabolomic profiling of diagnostic bone marrow plasma could provide insights into the underlying biology of early treatment response and inform treatment strategies for high-risk patients. METHODS: We performed global metabolomic profiling of samples from discovery (N = 93) and replication (N = 62) cohorts treated at Texas Children's Hospital. Next, we tested the cytotoxicity of drugs targeting central carbon metabolism in cell lines and patient-derived xenograft (PDX) cells. FINDINGS: Metabolite set enrichment analysis identified altered central carbon and amino acid metabolism in MRD-positive patients from both cohorts at a 5% false discovery rate. Metabolites from these pathways were used as inputs for unsupervised hierarchical clustering. Two distinct clusters were identified, which were independently associated with MRD after adjustment for immunophenotype, cytogenetics, and NCI risk group. Three nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which reduce glycolytic/TCA cycle activities, demonstrated nanomolar-range cytotoxicity in B- and T-ALL cell lines and PDX cells. INTERPRETATION: This study provides new insights into the role of central carbon metabolism in early treatment response and as a potential targetable pathway in high-risk disease. FUNDING: American Society of Hematology; Baylor College of Medicine Department of Paediatrics; Cancer Prevention and Research Institute of Texas; the Lynch family; St. Baldrick's Foundation with support from the Micaela's Army Foundation; United States National Institutes of Health.

Role of AMPK and PPARα in the anti-skin cancer effects of ursolic acid.

The phytonutrient ursolic acid (UA), present in apples, rosemary, and other plant sources, has anti-cancer properties in a number of systems, including skin cancers. However, few reports have examined upstream mechanisms by which UA may prevent or treat cancer. Recent reports have indicated UA induces death of cancer cell lines via AMP-activated protein kinase (AMPK), an energy-sensing kinase which possesses both pro-metabolic and anti-cancer effects. Other studies have shown UA activates peroxisome proliferator activated receptor α (PPARα) and the glucocorticoid receptor (GR). Here, we found the cytotoxic effect of UA in skin carcinoma cells required AMPK activation. In addition, two inhibitors of PPARα partially reversed the cytotoxic effects of UA, suggesting its effects are at least partially mediated through this receptor. Finally, inhibition of the GR did not reverse the effects of UA nor did this compound bind the GR under the conditions of experiments performed. Overall, studies elucidating the anti-cancer effects of UA may allow for the development of more potent analogues utilizing similar mechanisms. These studies may also reveal the mediators of any possible side effects or resistance mechanisms to UA therapy.

Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability.

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.

DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells.

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARα receptors.

Glucocorticoids (GCs) are well-known anti-inflammatory compounds, but they also inhibit cell proliferation depending on cell type. Similarly, peroxisome proliferator-activated receptors (PPARα, PPARδ, and PPARγ) also possess anti-proliferation properties beyond their canonical roles as metabolic mediators. In the present study, we investigated the potential additive or synergistic inhibitory effects on cancer cell proliferation by simultaneous application of fenofibrate and budesonide, agonists for PPARα and glucocorticoid receptor, respectively. We observed differential effects on cell proliferation in A549 and SK-MES-1 lung cancer cells by budesonide and fenofibrate. Fenofibrate inhibited cell proliferation in both TP53 wild type and deficient lung cancer cells. The anti-proliferation effect of budesonide in TP53 wild type A549 cells was abolished in SK-MES-1 cells that do not have wild type TP53 protein. An additive effect against cell proliferation by budesonide and fenofibrate combination was observed only in TP53 wild type A549 cancer cells. Analysis of cell cycle distribution and cyclin profile indicated that the inhibition of cell proliferation was associated with G1 cell cycle arrest. The suppression of NF-κB activity and ERK signaling may contribute to the inhibition of cell proliferation by budesonide and or fenofibrate. The additive inhibitory effect on cell proliferation by budesonide and fenofibrate combination suggests that the same or greater therapeutic effect could be achieved with reduced dosage and side effects when the two compounds are applied simultaneously.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice.

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Resveratrol and P-glycoprotein inhibitors enhance the anti-skin cancer effects of ursolic acid.

UNLABELLED: Ursolic acid, present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to ursolic acid treatment. Herein, skin carcinoma cells (Ca3/7) as compared with skin papilloma cells (MT1/2) displayed more resistance to ursolic acid-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in preclinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate rhodamine 123 (Rh123) indicating ursolic acid is transported by P-gp. To determine whether P-gp inhibition can enhance ursolic acid-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil or cyclosporin A. Alternatively, cells were pretreated with the natural compound resveratrol, a known chemotherapy sensitizer. Verapamil and resveratrol enhanced the effects of ursolic acid in both cell lines, whereas cyclosporin A only did so in Ca3/7 cells. Similarly, verapamil inhibited Rh123 efflux in both lines, whereas cyclosporin A only inhibited Rh123 efflux in Ca3/7 cells. Resveratrol did not inhibit Rh123 efflux in either line, indicating the synergistic effects of resveratrol and ursolic acid are not manifest by inhibition of P-gp-mediated efflux of ursolic acid. These results indicate that the anti-skin cancer effects of ursolic acid are enhanced with P-gp inhibitors. In addition, resveratrol and ursolic acid interact synergistically, but not through inhibition of P-gp. IMPLICATIONS: Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen.

Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice.

The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

The possible separation of 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation and hyperplasia by compound A.

Activated glucocorticoid receptor (GR) acts via two different mechanisms: transcriptional regulation that requires DNA-binding, and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1). It has been postulated that many important effects of glucocorticoids, including their anti-inflammatory properties, depend on GR's transrepressive effects on NF-κB and AP-1. In the present study, we have employed a TPA-induced model of skin inflammation and epidermal hyperplasia to determine whether partial activation of the glucocorticoid receptor by compound A (CpdA) is sufficient to reverse the effect of TPA treatment. CpdA is a nonsteroidal GR modulator with high binding affinity, is capable of partial activation of GR. Topical application of TPA twice per week for 2 wk results in inflammation and epidermal hyperplasia. TPA treatment also elevates levels of c-jun (AP-1 component), cyclooxygenase-2 (COX-2), p50 (NF-κB component), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in the skin. Fluocinolone acetonide (FA) (a full GR agonist) was able to completely reverse the above effects of TPA. When applied alone, CpdA increased the epidermal thickness and keratinocyte proliferation as well as levels of c-jun, COX-2, IL-6, and IFN-γ. However, CpdA treatment resulted in a decrease in the number of p50 positive cells induced by TPA, suggesting its role in inhibition of NF-κB. The level of metallothionein-1 mRNA, regulated by GR was also significantly decreased in skin samples treated with CpdA. Our results suggest that CpdA is able to inhibit GR transactivation and activate only some transrepression properties of GR.