Fanning the Flames of Endoplasmic Reticulum (ER) Stress: Can Sphingolipid Metabolism Be Targeted to Enhance ER Stress-Associated Immunogenic Cell Death in Cancer?

The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress. Given that sphingolipids have both prosurvival and proapoptotic effects, they also exert opposing effects on life/death decisions in the face of prolonged ER stress detected by the UPR. In this review, we will focus on several recent studies that demonstrate how sphingolipids affect each arm of the UPR. We will also discuss the role of sphingolipids in the process of immunogenic cell death downstream of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiating factor 2α (eIF2α) arm of the UPR. Furthermore, we will discuss strategies to target the sphingolipid metabolic pathway that could potentially act synergistically with agents that induce ER stress as novel anticancer treatments. SIGNIFICANCE STATEMENT: This review provides the readers with a brief discussion of the sphingolipid metabolic pathway and the unfolded protein response. The primary focus of the review is the mechanism(s) by which sphingolipids modulate the endoplasmic reticulum (ER) stress response pathways and the critical role of sphingolipids in the process of immunogenic cell death associated with the ER stress response.

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell death-promoting signaling lipid that plays a central role in therapy-induced cell death. Acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug, LCL-805, across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

Characterization of Anticancer Effects of the Analogs of DJ4, a Novel Selective Inhibitor of ROCK and MRCK Kinases.

The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKß) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/ß. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells. To further optimize the ROCK/MRCK inhibitory potency of DJ4, we generated a library of 27 analogs. Among the various structural modifications, we identified four additional active analogs with enhanced ROCK/MRCK inhibitory potency. The anti-proliferative and cell cycle inhibitory effects of the active analogs were examined in non-small cell lung cancer, breast cancer, and melanoma cell lines. The anti-proliferative effectiveness of DJ4 and the active analogs was further demonstrated against a wide array of cancer cell types using the NCI-60 human cancer cell line panel. Lastly, these new analogs were tested for anti-migratory effects in highly invasive MDA-MB-231 breast cancer cells. Together, our results demonstrate that selective inhibitors of ROCK1/2 (DJE4, DJ-Allyl) inhibited cell proliferation and induced cell cycle arrest at G2/M but were less effective in cell death induction compared with dual ROCK1/2 and MRCKα/ß (DJ4 and DJ110).

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNß and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells.

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ12-PGJ2, and 15d-PGJ2, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44-/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.

Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes.

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3ß, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

IGF2BP1 regulates the cargo of extracellular vesicles and promotes neuroblastoma metastasis.

Neuroblastoma is a highly metastatic cancer, and thus is one of the leading causes of cancer-related mortalities in pediatric patients. More than 50% of NB cases exhibit 17q21-ter partial chromosomal gain, which is independently associated with poor survival, suggesting the clinical importance of genes at this locus in NB. IGF2BP1 is one such proto-oncogene located at 17q locus, and was found to be upregulated in patients with metastatic NBs. Here, utilizing multiple immunocompetent mouse models, along with our newly developed highly metastatic NB cell line, we demonstrate the role of IGF2BP1 in promoting NB metastasis. Importantly, we show the significance of small extracellular vesicles (EVs) in NB progression, and determine the pro-metastatic function of IGF2BP1 by regulating the NB-EV-protein cargo. Through unbiased proteomic analysis of EVs, we discovered two novel targets (SEMA3A and SHMT2) of IGF2BP1, and reveal the mechanism of IGF2BP1 in NB metastasis. We demonstrate that IGF2BP1 directly binds and governs the expression of SEMA3A/SHMT2 in NB cells, thereby modulating their protein levels in NB-EVs. IGF2BP1-affected levels of SEMA3A and SHMT2 in the EVs, regulate the formation of pro-metastatic microenvironment at potential metastatic organs. Finally, higher levels of SEMA3A/SHMT2 proteins in the EVs derived from NB-PDX models indicate the clinical significance of the two proteins and IGF2BP1-SEMA3A/SHMT2 axis in NB metastasis.

Signaling pathways and regulation of gene expression in hematopoietic cells.

Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins. Protein kinases and phosphatases regulate gene expression by reversible phosphorylation of transcriptional factors, which are their direct substrates. Casein kinase II (CK2) is a serine/threonine kinase that phosphorylates a large number of proteins that have critical roles in cellular proliferation, metabolism and survival. Altered function of CK2 has been associated with malignant transformation, immunological disorders and other types of diseases. Protein phosphatase 1 (PP1) is a serine/threonine phosphatase, which regulates the phosphorylation status of many proteins that are essential for cellular functions. IKAROS is a DNA-binding protein, which functions as a regulator of gene transcription in hematopoietic cells. CK2 directly phosphorylates IKAROS at multiple phosphosites which determines IKAROS activity as a regulator of gene expression. PP1 binds to IKAROS via the PP1-consensus recognition site and dephosphorylates serine/threonine residues that are phosphorylated by CK2. Thus, the interplay between CK2 and PP1 signaling pathways have opposing effects on the phosphorylation status of their mutual substrate - IKAROS. This review summarizes the effects of CK2 and PP1 on IKAROS role in regulation of gene expression and its function as a tumor suppressor in leukemia.

A Descriptive Study of Some of the Dento-Gingival Components of Esthetics of Patients at a Tertiary Care-center in the Eastern Part of Nepal.

Background: The purpose of this study was to observe and describe some of the dento-gingival components of esthetics like the mean gingival zenith position (GZP) with respect to the vertical bisected midline axis (VBM), relative gingival zenith level (GZL) of lateral incisors (LIs), heights and widths of central incisors (CIs), LIs, and their ratios. Materials and Methods: This cross-sectional, hospital-based, descriptive study was conducted from Feb 2019 to Aug 2019. Ethical clearance was taken from the Institutional Review Committee of B. P. Koirala Institute of Health Sciences. Convenience sampling was done. Variables (above mentioned) were marked and measured on the casts with a calibrated digital vernier caliper and entered in the data sheet, and descriptive analysis was done with SPSS version 20. Results: A total of 210 subjects of the age group 18-25 years were included in the study. Mean GZP of the right CI was 0.49 ± 0.54 mm, of the left CI was 0.42 ± 0.43 mm, of the right LI was 0.20 ± 0.34 mm, of the left LI was 0.04 ± 0.35 mm, of the right C (Canine) was 0.023 ± 0.38 mm, and of left C was 0.07 ± 0.38 mm. Mean relative GZL of LI was about 0.40-0.41 mm below the reference line. Mean height of the right CI was 9.34 ± 0.93 mm, and that of the left CI was 9.31 ± 0.87 mm; that of the right LI was 7.91 ± 0.98 mm, and that of the left LI was 7.92 ± 0.91 mm. Mean width of the right CI was 8.34 ± 0.57 mm, and that of the left CI was 8.38 ± 0.48 mm; that of the right LI was 6.62 ± 0.58 mm, and that of the left LI was 6.66 ± 0.53 mm. Conclusions: Mean GZP of each upper anterior tooth was distally located with respect to VBM; that of CI was more distally placed than LI and C. Relative GZL of LI was below the reference line. The central incisor width/height ratio obtained was >80% that means a squarer tooth.

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Therapeutic Benefits of Selenium in Hematological Malignancies.

Supplementing chemotherapy and radiotherapy with selenium has been shown to have benefits against various cancers. This approach has also been shown to alleviate the side effects associated with standard cancer therapies and improve the quality of life in patients. In addition, selenium levels in patients have been correlated with various cancers and have served as a diagnostic marker to track the efficiency of treatments or to determine whether these selenium levels cause or are a result of the disease. This concise review presents a survey of the selenium-based literature, with a focus on hematological malignancies, to demonstrate the significant impact of selenium in different cancers. The anti-cancer mechanisms and signaling pathways regulated by selenium, which impart its efficacious properties, are discussed. An outlook into the relationship between selenium and cancer is highlighted to guide future cancer therapy development.

Interleukin-4 treatment reduces leukemia burden in acute myeloid leukemia.

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response, particularly in allergy and hypersensitivity. Interestingly, IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites, Δ12 -prostaglandin J2 (Δ12 -PGJ2 ) and 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ), collectively called cyclopentenone PGs (CyPGs). However, the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here, we employed a murine model of acute myeloid leukemia (AML), where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells, to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML, as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated, in part, by the enhanced expression of hematopoietic- PGD2  synthase (H-PGDS) to effect endogenous production of CyPGs, through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662, a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, suggested endogenous CyPGs-PPARγ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together, our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML.

Harnessing the power of sphingolipids: Prospects for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.

Acute Myeloid Leukemia Stem Cells: Origin, Characteristics, and Clinical Implications.

The stem cells of acute myeloid leukemia (AML) are the malignancy initiating cells whose survival ultimately drives growth of these lethal diseases. Here we review leukemia stem cell (LSC) biology, particularly as it relates to the very heterogeneous nature of AML and to its high disease relapse rate. Leukemia ontogeny is presented, and the defining functional and phenotypic features of LSCs are explored. Surface and metabolic phenotypes of these cells are described, particularly those that allow distinction from features of normal hematopoietic stem cells (HSCs). Opportunities for use of this information for improving therapy for this challenging group of diseases is highlighted, and we explore the clinical needs which may be addressed by emerging LSC data. Finally, we discuss current gaps in the scientific understanding of LSCs.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting FAS signaling.

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, whereas long noncoding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between HOTTIP lncRNA and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-seq (HOTTIP binding in genomewide), ChIP-seq, and ATAC-seq, we found that some miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a, and miR-10b), located cis and trans, were most dramatically regulated and significantly decreased in HOTTIP-/- AML cells. HOTTIP bound to the miR-196b promoter and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster-associated miRNAs in AML cells, whereas reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival of mice compared to wild-type cells transplanted into mice. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription and consequently represses tumor suppressors and promotes leukemogenesis.

DJ4 Targets the Rho-Associated Protein Kinase Pathway and Attenuates Disease Progression in Preclinical Murine Models of Acute Myeloid Leukemia.

The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2-5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05-1.68 µM) and primary patient cells (IC50: 0.264-13.43 µM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 µM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy.

Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia.

Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS' transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL (B cell Lymphoma like-2 like 1, BCL2L1). CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy.