Search

Diamond-Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because more than 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is due predominantly to a block and delay in early committed erythropoiesis with reduced megakaryocyte/erythroid progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA sequencing on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19 insufficiency. However, SATB1 expression did not affect expansion of committed erythroid progenitors, indicating ribosomal insufficiency affects multiple stages during erythroid differentiation.

Supplementary Figure 3 from The Multitargeted Receptor Tyrosine Kinase Inhibitor Linifanib (ABT-869) Induces Apoptosis through an Akt and Glycogen Synthase Kinase 3β–Dependent Pathway

Supplementary Figure 1 from The Multitargeted Receptor Tyrosine Kinase Inhibitor Linifanib (ABT-869) Induces Apoptosis through an Akt and Glycogen Synthase Kinase 3β–Dependent Pathway

Supplementary Figure 2 from The Multitargeted Receptor Tyrosine Kinase Inhibitor Linifanib (ABT-869) Induces Apoptosis through an Akt and Glycogen Synthase Kinase 3β–Dependent Pathway

The FMS-like receptor tyrosine kinase 3 (FLT3) plays an important role in controlling differentiation and proliferation of hematopoietic cells. Activating mutations in FLT3 occur in patients with acute myeloid leukemia (AML; 15%–35%), resulting in abnormal cell proliferation. Furthermore, both adult and pediatric patients with AML harboring the FLT3 internal tandem duplication (ITD) mutation have a poor prognosis. Several inhibitors have been developed to target mutant FLT3 for the treatment of AML, yet the molecular pathways affected by drug inhibition of the mutated FLT3 receptor alone have not been characterized as yet. Linifanib (ABT-869) is a multitargeted tyrosine kinase receptor inhibitor that suppresses FLT3 signaling. In this article, we show that treatment with linifanib inhibits proliferation and induces apoptosis in ITD mutant cells in vitro and in vivo. We show that treatment with linifanib reduces phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β). In addition, we show that inhibition of GSK3β decreases linifanib-induced apoptosis. This study shows the importance of GSK3 as a potential target for AML therapy, particularly in patients with FLT3 ITD mutations. Mol Cancer Ther; 10(6); 949–59. ©2011 AACR.

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.Significance:Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg. Cancer Res; 78(22); 6497–508. ©2018 AACR.

Unbiased shRNA library screening and global transcriptome analyses reveal mechanisms that modulate dasatinib sensitivity and suggest therapeutic strategies to improve outcome of patients with acute lymphoblastic leukemia.

Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT gene rearrangement. Supplementary Fig. S2. Effects of dinaciclib on genome-wide chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S3: C-Myc and MYCN protein expression in four NB cell lines. Supplementary Fig. S4: Effects of dinaciclib on the chromatin occupancy of epigenetic modulators at Dvl2, AURKA, and AURKB and in CLB-GA cells. Supplementary Fig. S5. Association of overexpression of FEN1, BIRC5, and UHRF1 with poor prognosis and effects of dinaciclib on chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S6. JQ1 sensitive Brd4 binding profiles in OCI-AML3 cells. Supplementary Fig. S7. JQ1 and dinaciclib are synergistic or additive in inducing cytotoxicity in NB cell lines. Supplementary Fig. S8. Combination index (CI) values for synergy experiment combining Dinaciclib with Brd4 inhibitor JQ1. Supplementary Fig. S9. Effects of dinaciclib on phosphorylation of Rb in CLB-GA and Kelly cells. Supplementary table S1. Primer sequences used in ChIP-qPCR and real-time RT-PCR analysis. Supplementary table 2. Gene sets enriched in NB patients with TERT gene rearrangement (n=32) versus without TERT gene rearrangement (n=278) within MYCN non-amplified NB of Tumor Neuroblastoma (TERT)-Fischer-394-custom. Supplementary table 3. Gene sets enriched in NB patients with high versus low TERT within Tumor Neuroblastoma Gene-TARGET-161. Supplementary table 4: Information on patient samples used in this study.

Correction: Cancer Res 2008;68:2557-60 from Role of the Aggresome Pathway in Cancer: Targeting Histone Deacetylase 6–Dependent Protein Degradation

Misfolded or aggregated proteins have two fates: they are either refolded with the help of chaperones or degraded by the proteasome. Cells also have an alternative pathway that involves intracellular “storage bins” for misfolded intracellular proteins known as aggresomes. Aggresomes recruit motor proteins that transport misfolded or aggregated proteins to chaperones and proteasomes for subsequent destruction. There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the role of aggresomes in cancer and the potential to target this pathway for therapy. [Cancer Res 2008;68(8):2557–60]

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker.ImportanceHuman exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

Chronic myeloid leukemia (CML) is effectively treated with long-term tyrosine kinase inhibitor (TKI) therapy, yet little is known about risks of prolonged TKI exposure in young patients, and long-term effect monitoring is not standardized. We surveyed North American pediatric oncologists (n = 119) to evaluate perceived risk of and surveillance practices for potential toxicities associated with prolonged TKI exposure in children and adolescents/young adults (AYAs) with CML. Survey domains included general and specific risk perceptions and surveillance practices for asymptomatic patients on chronic TKI therapy. We analyzed data descriptively and explored relationships between risk perceptions and surveillance. Risk perceptions varied among oncologists but were similar across six categories (thyroid, cardiac, vascular, metabolic, fertility, psychologic), with less than one-third rating each risk as moderate or high in pediatric and AYA patients. More oncologists perceived moderate or high risk of growth abnormalities in children (62% pediatric, 14% AYA) and financial toxicity in all patients (60% pediatric, 64% AYA). A greater proportion of oncologists with moderate or high perceived risk of thyroid abnormalities reported testing thyroid function compared to those with lower perceived risk; patterns for metabolic risk/lipid tests and cardiac risk/tests were similar. In summary, we found that pediatric oncologists had variable risk perceptions and surveillance practices for potential toxicities associated with prolonged TKI exposure. Standardizing surveillance would help quantify risks and refine recommendations. Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2021.2017085 .

B- and T-cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anticancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells from high-risk patients with B/T-ALL using mass cytometry, flow cytometry, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining acute lymphoblastic leukemia (ALL) regression. We found that, compared with their normal counterparts, NK cells from patients with B/T-ALL are less cytotoxic but exhibit an activated signature that is characterized by high CD56, high CD69, production of activated NK cell–origin cytokines, and calcium (Ca2+) signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells from ALL from lysing NK cell–sensitive targets as efficiently as do normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in patients with ALL. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK cell infusions to prevent ALL recurrence.

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy. Significance: Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

Ribosomal S6 Kinases (RSKs) are a group of serine/threonine kinases that function downstream of the Ras/Raf/MEK/ERK signaling pathway. Four RSK isoforms are directly activated by ERK1/2 in response to extracellular stimuli including growth factors, hormones, and chemokines. RSKs phosphorylate many cytosolic and nuclear targets resulting in the regulation of diverse cellular processes such as cell proliferation, survival, and motility. In hematological malignancies such as acute myeloid leukemia (AML), RSK isoforms are highly expressed and aberrantly activated resulting in poor outcomes and resistance to chemotherapy. Therefore, understanding RSK function in leukemia could lead to promising therapeutic strategies. This review summarizes the current information on human RSK isoforms and discusses their potential roles in the pathogenesis of AML and mechanism of pharmacological inhibitors.

Background Targeted anticancer therapies such as BCR-ABL tyrosine kinase inhibitors (TKIs) have improved outcomes for chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). However, little is known about long-term risks of TKIs in children. Exposure-based survivorship guidelines do not include TKIs, thus surveillance practices may be variable. Methods We retrospectively examined surveillance for cardiac and endocrine late effects in children receiving TKIs for Ph + leukemias, diagnosed at Results 66 patients (CML n = 44; Ph + ALL n = 22) met inclusion criteria. Among patients with CML, ≥1 evaluation was done: ECHO (50.0%), EKG (48.8%), TSH (43.9%), DXA (2.6%), bone age (7.4%). Among patients with Ph + ALL, ≥1 evaluation was done: ECHO (86.4%), EKG (68.2%), TSH (59.1%), DXA (63.6%), bone age (44.4%). Over a median 6.3 and 5.7 years of observation, respectively, 2% of patients with CML and 57% with Ph + ALL attended a survivorship clinic. Conclusions Despite common exposure to TKIs in survivors of Ph + leukemias, patterns of surveillance for late effects differed in CML and Ph + ALL, with the latter receiving more surveillance likely due to concomitant chemotherapy exposures. Targeted therapies such as TKIs are revolutionizing cancer treatment, but surveillance for late effects and referral to survivorship clinics are variable despite the chronicity of exposure. Evidence based guidelines and longer follow-up are needed.

Chronic myelogenous leukemia (CML) is rare in children, requiring extrapolation from treatment of adults. In this review, we explore similarities and differences between adult and pediatric CML with a focus on therapeutic advances and emerging clinical questions. Pediatric CML is effectively treated with long-term targeted therapy using tyrosine kinase inhibitors (TKIs). Newly diagnosed pediatric patients in chronic phase can now be treated with imatinib, dasatinib, or nilotinib without allogeneic hematopoietic stem cell transplantation. While treatment-free remission is possible in adults in chronic phase with optimal response to therapy, data are currently insufficient to support stopping TKI in pediatrics outside of a clinical trial. Knowledge gaps remain regarding long-term and late effects of TKIs in pediatric CML. Targeted therapy has markedly improved outcomes for pediatric CML, while raising a number of clinical questions, including the possibility of treatment-free remission and long-term health implications of prolonged TKI exposure at a young age.

Chronic myelogenous leukemia (CML) is rare in children, requiring extrapolation from treatment of adults. In this review, we explore similarities and differences between adult and pediatric CML with a focus on therapeutic advances and emerging clinical questions.Pediatric CML is effectively treated with long-term targeted therapy using tyrosine kinase inhibitors (TKIs). Newly diagnosed pediatric patients in chronic phase can now be treated with imatinib, dasatinib, or nilotinib without allogeneic hematopoietic stem cell transplantation. While treatment-free remission is possible in adults in chronic phase with optimal response to therapy, data are currently insufficient to support stopping TKI in pediatrics outside of a clinical trial. Knowledge gaps remain regarding long-term and late effects of TKIs in pediatric CML. Targeted therapy has markedly improved outcomes for pediatric CML, while raising a number of clinical questions, including the possibility of treatment-free remission and long-term health implications of prolonged TKI exposure at a young age.

This chapter focuses on the development of therapeutics that work to induce the degradation of nuclear receptors. These therapeutics, unlike traditional small molecule inhibitors, work by redirecting the cellular waste disposal machinery to remove a therapeutic target. They are advantageous because they work through a catalytic mechanism, raising the possibility of drugs with enhanced potency. Current work in the field is toward identifying novel molecular “glues,” in the hopes of bringing proteolysis targeting chimeras (PROTACs) to the clinic. However, translating it from a concept dreamed up in a laboratory to a clinically viable tool took almost 20 years of basic research.

Diamond–Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3′ untranslated region (3′UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

Our concept of cancer latency, the interval from when a cancer starts until it is diagnosed, has changed dramatically. A prior widely-used definition was the interval between an exposure to a cancer-causing substance and cancer diagnosis. However, this definition does not accurately reflect current knowledge of how most cancers develop assuming, mostly incorrectly, one exposure is the sole cause of a cancer, ignoring the possibility the cancer being considered would have developed anyway but that the exposure accelerated cancer development and eliding the randomness in when a cancer is diagnosed. We show, using chronic myeloid leukaemia as a model, that defining cancer latency is not as simple as it once seemed. It is difficult or impossible to know at which event or mutation to start to clock to measure cancer latency. It is equally difficult to know when to stop the clock given the stochastic nature of when cancers are diagnosed. Importantly, even in genetically-identical twins with the same driver mutation intervals to develop cancer vary substantially. And we discuss other confonders. Clearly we need a new definition of cancer latency or we need to abandon the concept of cancer latency in the modern era of cancer biology.

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

A molecular systems architecture is presented for acute myeloid leukemia (AML) to provide a framework for organizing the complexity of biomolecular interactions. AML is a multifactorial disease resulting from impaired differentiation and increased proliferation of hematopoietic precursor cells involving genetic mutations, signaling pathways related to the cancer cell genetics, and molecular interactions between the cancer cell and the tumor microenvironment, including endothelial cells, fibroblasts, myeloid-derived suppressor cells, bone marrow stromal cells, and immune cells (e.g., T-regs, T-helper 1 cells, T-helper 17 cells, T-effector cells, natural killer cells, and dendritic cells). This molecular systems architecture provides a layered understanding of intra- and inter-cellular interactions in the AML cancer cell and the cells in the stromal microenvironment. The molecular systems architecture may be utilized for target identification and the discovery of single and combination therapeutics and strategies to treat AML.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL. Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg. Cancer Res; 78(22); 6497–508. ©2018 AACR.

Simple Summary To investigate whether pediatric and adult chronic myeloid leukemia (CML) have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML cells using high-throughput RNA sequencing. We identified differentially expressed genes and pathways unique to pediatric CML cells compared to adult CML cells. The Rho pathway was significantly dysregulated in pediatric CML cells compared to adult CML cells, suggesting the potential importance in the pathogenesis of pediatric CML. Our study is the first to compare transcriptome profiles of CML across different age groups. A better understanding of the biology of CML across different ages may inform future treatment approaches. Abstract Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

Diamond Blackfan Anemia (DBA) is associated with a hypoproliferative anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life with the majority of patients carrying mutations in one of at least 17 ribosomal proteins, with RPS19 being the most common. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions, chronic effects from long term corticosteroid use, or complications from stem cell transplantation. The serine threonine kinase Nemo-like Kinase (NLK) is an atypical member of the MAP kinase family of enzymes and has been shown to be chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as in erythroid progenitors from DBA patients. In RPS19-insufficient human hematopoietic stem and progenitor cells, genetic silencing of NLK by shRNA increased erythroid expansion by 220.3% (SD = 6.6%), indicating that aberrant NLK activation may contribute to the pathogenesis of the disease and is a potential target for DBA therapy. A number of clinically approved or advanced compounds have been developed to inhibit MAP kinases with various degrees of cross reactivity among its family members. We therefore screened a number of compounds that inhibit NLK as an off-target and found that these NLK inhibitors improved erythroid expansion in DBA models. Of these inhibitors, OTS-167 performed optimally, improving erythropoiesis by 2-fold at 300nM, with an EC50 of 146nM. Previous studies of OTS-167 in xenograft models of neuroblastoma for one month did not result in neutropenia, suggesting very little to no toxicity to myeloid cells. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or treatment with steroids. Our results suggest that pharmacologic inhibition of NLK is a potential approach to treat patients with DBA. We are currently investigating other NLK inhibitors in preclinical models for future clinical application. Disclosures Glader: Agios: Consultancy.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease. We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA. Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated. Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors. Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31). Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. Disclosures Glader: Agios: Consultancy.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric cancer characterized by excessive accumulation of monocytes/macrophages with myelodysplastic features and splenomegaly. Hematopoietic stem cell transplantation (HSCT) is the only curative option, but relapse occurs in ~50% of the patients. Mutations in the RAS pathway are the major drivers and DNA hypermethylation is associated with severe clinical phenotype. DNA methylation is linked to histone modifications in normal cell development and cancer pathogenesis. Gene expression levels are programmed by histone modifications. However, a comprehensive characterization of histone modifications in JMML is yet to be elucidated. It was reported that the CD34+ hematopoietic stem and progenitors cells (HSPCs) possess leukemia initiation potential (Caye et al., 2015; Krombholz et al., 2016; Louka et al., 2021; Yoshimi et al., 2017). We hypothesized that dysregulated histone modifications contribute to leukemogenesis in JMML. To this goal, we investigated epigenetic landscape at the single-cell level in JMML HSPCs using cytometry by time-of-flight (EpiTOF). We isolated HSPCs from the spleens of JMML patients with PTPN11 mutations which were collected at diagnosis, before treatment (provided by the European Working Groups of Myelodysplastic Syndromes). The CD34+ cells from JMML Spleens (n=5) and Bone Marrow (BM n=1) alongside healthy controls (cord blood CB n=5; BM n=2) were stained with antibodies against 15 immunophenotypic and 46 key histone markers and subsequently analyzed on a mass cytometer (Fluidigm, Helios). We found that a group of key histone markers were significantly downregulated in JMML in comparison to healthy CBs, including H4K16ac, H3K23ac, H3K56ac, H3K36me1, H3K9me1 and H3K27me2 (Fig.1). Dimensionality reduction analyses showed a global reduction in histone acetylation in all JMML spleen samples (~70%) when compared to CBs (average >95%). Our study revealed significant dysregulation of key histone markers and their respective modifiers (histone modifying enzymes) within the leukemogenic CD34+ compartment (Fig.2). Our findings demonstrated an aberrant epigenetic regulation in JMML patient samples with concurrent loss of histone acetylation and some histone methylation markers, which have been reported as hallmarks of other cancers. Further characterization of the epigenetic landscape in JMML was performed using transposase-accessible chromatin with high-throughput sequencing (bulk ATAC-seq). Notably, ATAC-seq data revealed significantly lower expression of key histone acetyltranferases (HAT), such as Kat6b and Kat8 (Fig.2) , which specifically regulate acetylation of H3K23 and H4K16 respectively. Additionally, an increased activity of histone deacetylase, HDAC9, was also observed in these samples (Fig.2). Collectively, our data suggests aberrant activities of histone modifying enzymes (HMEs) in JMML HSPCs (Fig.2), with both increased HDAC and decreased HAT activities observed in our ATAC-data. This finding is concurrent with our EpiTOF findings of global loss of histone acetylation in JMML (Fig.1A). It is known that histone modifications are easier to reverse when compared to DNA methylation, and we believe targeted inhibition of specific HDACs that we have found to be upregulated in JMML (such as HDAC9) might hold great therapeutic potential. Additionally, investigation into the impact of altered chromatin structure of JMML HSPCs on gene expression is currently ongoing using single-cell RNA-seq. In conclusion, our study identified a novel mechanism of epigenetic dysregulation in putative JMML leukemia initiating cells related to imbalanced activities of histone modifying enzymes which could potentially represent future therapeutic targets. Figure 1 Figure 1. Disclosures Bertaina: AdicetBio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees.

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.