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Asparaginase is a key component of pediatric-inspired regimens in young adults with acute lymphoblastic leukemia (ALL). Truncation of asparaginase therapy is linked to inferior outcomes in children with ALL. However, a similar correlation in adults is lacking. Here, we studied the prevalence and risk factors associated with pegylated (PEG)-asparaginase discontinuation in young adults with ALL treated on the US intergroup Cancer and Leukemia Group B (CALGB) 10403 study and examined the prognostic impact of early discontinuation (ED) (defined as

Figure S1. SACLAC reduces AC activity at lower doses than LCL204. Figure S2. SACLAC treatment increases production of multiple ceramide species in human AML cell lines. Figure S3. SACLAC is more toxic to AML cells than normal cells. Figure S4. SACLAC mechanism of action is consistent in multiple human AML cell lines. Figure S5. Exogenous ceramides induce apoptosis in human AML cell lines. Figure S6. AC knockdown alters splicing of Mcl-1 and Bim in HL-60/VCR cells via regulation of SF3B1. Figure S7. Baseline apoptosis varies based on type of electroporated content. Figure S8. Proposed model of SACLAC mechanism of action. Figure S9. IV administration of SACLAC improves delivery and lipids are shifted in mice.

Supplemental Table 1: Analysis of potential confounding factors; Supplemental Table 2: Multivariate Cox proportional hazard analysis; Supplemental Figure 1: The expression of CXCR5 and TCF1; Supplemental Figure 2: Comparing Fraction III CD8 T cells vs. overall survival; Supplemental Figure 3: The expression of CD39.

TIGIT expression associates with up-regulation of multiple inhibitory receptors.

Legends for supplemental figures

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Immunotherapy targeting inhibitory pathways to unleash the antileukemia T-cell response is a promising strategy for the treatment of leukemia, but we must first understand the underlying molecular mechanisms. Eomesodermin (Eomes) and T-bet are both T-box transcription factors that regulate CD8+ T-cell responses in a context-specific manner. Here, we examined the role of these transcription factors in CD8+ T-cell immunity in AML patients. We report that the frequency of Eomes+T-betlow CD8+ T cells increased in newly diagnosed AML. This cell subset produced fewer cytokines and displayed reduced killing capacity, whereas depletion of Eomes by siRNA reversed these functional defects. Furthermore, Eomes bound the promoter of T-cell immunoglobulin and ITIM domain (TIGIT) and positively regulated the expression of this inhibitory receptor on patient-derived T cells. A high frequency of Eomes+T-betlow CD8+ T cells was associated with poor response to induction chemotherapy and shorter overall survival in AML patients. These findings have significant clinical implications as they not only identify a predictive and prognostic biomarker for AML, but they also provide an important target for effective leukemia therapeutics.Significance:These findings reveal that a high frequency of Eomes+T-betlow CD8+ T cells predicts poor clinical outcome in AML and that targeting Eomes may provide a therapeutic benefit against AML.

TIGIT+ CD8+ T cells from AML patients retain capacity of proliferation and cytotoxicity.

Purpose: T-cell immunoglobulin and immunoreceptor tyrosine–based inhibitory motif (ITIM) domain (TIGIT) is a recently identified T-cell coinhibitory receptor. In this study, we aimed to determine the clinical impact of TIGIT in patients with acute myelogenous leukemia (AML) and dissect the role of TIGIT in the pathogenesis of leukemia progression.Experimental Design: TIGIT expression on T cells from peripheral blood collected from patients with AML was examined by flow cytometry. The correlation of TIGIT expression to clinical outcomes, including rate of complete remission and relapse post-allogeneic stem cell transplantation (alloSCT) in AML patients, was analyzed. Phenotypic and functional study (cytokine release, proliferation, killing, and apoptosis) of TIGIT-expressing T cells were performed. Using siRNA to silence TIGIT, we further elucidated the regulatory role of TIGIT in the T-cell immune response by dissecting the effect of TIGIT knockdown on cytokine release and apoptosis of T cells from AML patients.Results: TIGIT expression on CD8+ T cells is elevated in AML patients and high-TIGIT correlates with primary refractory disease and leukemia relapse post-alloSCT. TIGIT+ CD8+ T cells display phenotypic features of exhaustion and exhibit functional impairment manifested by low production of cytokines and high susceptibility to apoptosis. Importantly, their functional defects are reversed by TIGIT knockdown.Conclusions: TIGIT contributes to functional T-cell impairment and associates with poor clinical outcome in AML. Our study suggests that blockade of TIGIT to restore T-cell function and antitumor immunity may represent a novel effective leukemia therapeutic. Clin Cancer Res; 22(12); 3057–66. ©2016 AACR.

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.

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments.

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 D(2) (PGD(2)) and its metabolites, Δ(12)-prostaglandin J(2) (Δ(12)-PGJ(2)) and 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), 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- PGD(2) 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 AML patients.

Adolescents and young adults (AYAs) with acute lymphoblastic leukemia have improved outcomes when treated with pediatric-inspired regimens. CALGB 10403 was the largest prospective study to evaluate the feasibility of using a pediatric regimen in AYAs with acute lymphoblastic leukemia up to 40 years of age. This article presents the toxicity events observed in the CALGB 10403 study and compares these toxicities vs those observed among AYAs treated on the same arm of the companion Children’s Oncology Group (COG) AALL0232 study. Toxicities in CALGB 10403 were similar to those observed in COG AALL0232. Some grade 3 to 4 adverse events were more often reported in CALGB 10403 compared with COG AALL0232 (hyperglycemia, hyperbilirubinemia, transaminase elevation, and febrile neutropenia). Adverse events correlated with body mass index ≥30 kg/m2 and some with increasing age. The mortality rate in CALGB 10403 was low (4%) and similar to that in the COG AALL0232 trial. A caveat to this analysis is that only 39% of CALGB 10403 patients completed all planned protocol treatment. In COG AALL0232, although 74% of patients aged

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.

PURPOSE: Patients with acute myeloid leukemia with high-risk cytogenetics in first complete remission (CR1) achieve better outcomes if they undergo allogeneic hematopoietic cell transplantation (HCT) compared with consolidation chemotherapy alone. However, only approximately 40% of such patients typically proceed to HCT. METHODS: We used a prospective organized approach to rapidly identify donors to improve the allogeneic HCT rate in adults with high-risk acute myeloid leukemia in CR1. Newly diagnosed patients had cytogenetics obtained at enrollment, and those with high-risk cytogenetics underwent expedited HLA typing and were encouraged to be referred for consultation with a transplantation team with the goal of conducting an allogeneic HCT in CR1. RESULTS: Of 738 eligible patients (median age, 49 years; range, 18-60 years of age), 159 (22%) had high-risk cytogenetics and 107 of these patients (67%) achieved CR1. Seventy (65%) of the high-risk patients underwent transplantation in CR1 ( P < .001 compared with the historical rate of 40%). Median time to HCT from CR1 was 77 days (range, 20-356 days). In landmark analysis, overall survival (OS) among patients who underwent transplantation was significantly better compared with that of patients who did not undergo transplantation (2-year OS, 48% v 35%, respectively [ P = .031]). Median relapse-free survival after transplantation in the high-risk cohort who underwent transplantation in CR1 (n = 70) was 11.5 months (range, 4-47 months), and median OS after transplantation was 14 months (range, 4-44 months). CONCLUSION: Early cytogenetic testing with an organized effort to identify a suitable allogeneic HCT donor led to a CR1 transplantation rate of 65% in the high-risk group, which, in turn, led to an improvement in OS when compared with the OS of patients who did not undergo transplantation.

Summary Decitabine is a DNA‐hypomethylating agent that has been widely applied for the treatment of acute myeloid leukaemia (AML) patients who are elderly or unfit for intensive therapy. Although effective, the complete response rate to decitabine is only around 30% and the overall survival remains poor. Emerging data support that regulation of DNA methylation is critical to control immune cell development, differentiation and activation. We hypothesize that defining how decitabine influences the immune responses in AML will facilitate the development of novel immune‐based leukaemia therapeutics. Here, we performed phenotypic and functional immune analysis on clinical samples from AML patients receiving decitabine treatment and demonstrated a significant impact of decitabine on the immune system. T‐cell expression of inhibitory molecules was upregulated and the ability of CD8 T cells to produce cytokines was decreased upon decitabine treatment. Importantly, in an unbiased comprehensive analysis, we identified a unique immune signature containing a cluster of key immune markers that clearly separate patients who achieved complete remission after decitabine from those who failed to do so. Therefore, this immune signature has a strong predictive value for clinical response. Collectively, our study suggests that immune‐based analyses may predict clinical response to decitabine and provide a therapeutic strategy to improve the treatment of AML.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. Vinca alkaloids such as vincristine (VCR), widely used in cancer treatment, are no exception, as their beneficial actions are often supplanted by resistance. In the present work we utilized HL-60 human leukemia cells and a VCR-resistant counterpart, HL-60/VCR, as a model to determine potential interplay between SL metabolism and mitochondrial bioenergetics supportive of multidrug resistance (MDR). Relative to wild-type cells, HL-60/VCR presented with global alterations in SL composition, typified by upregulated expression of sphingosine kinase (SPHK1), which catalyzes formation of sphingosine 1-phosphate (S1P), glucosylceramide synthase (GCS), which catalyzes formation of glucosylceramides (GC), and acid ceramidase, responsible for ceramide hydrolysis. In support of these changes, VCR resistance was also characterized by increases in S1P, several molecular species of ceramide and GC, and changes in sphingomyelin (SM) molecular species. With respect to mitochondria, despite increased basal respiration in HL-60/VCR cells, direct interrogation of the mitochondrial network revealed intrinsic respiratory complex insufficiency, largely localized to complex I (CI). Importantly, forced ceramide accumulation in wild-type cells phenocopied the respiratory insufficiency observed in HL-60/VCR, and co-targeting SL metabolism and CI induced synergistic cytotoxicity in HL-60/VCR cells, as well as in other MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

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.

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.

BackgroundAcute myeloid leukemia (AML) is fatal in elderly patients who are unfit for standard induction chemotherapy. The objective of this study was to evaluate the survival benefit of administering sapacitabine, an oral nucleoside analogue, in alternating cycles with decitabine, a low-intensity therapy, to elderly patients with newly diagnosed AML.MethodsThis randomized, open-label, phase 3 study (SEAMLESS) was conducted at 87 sites in 11 countries. Patients aged ≥70 years who were not candidates for or chose not to receive standard induction chemotherapy were randomized 1:1 to arm A (decitabine in alternating cycles with sapacitabine) received 1-hour intravenous infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 8 weeks (first cycle and subsequent odd cycles) and sapacitabine 300 mg twice daily on 3 consecutive days per week for 2 weeks every 8 weeks (second cycle and subsequent even cycles) or to control arm C who received 1-hour infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 4 weeks. Prior hypomethylating agent therapy for preexisting myelodysplastic syndromes or myeloproliferative neoplasms was an exclusion criterion. Randomization was stratified by antecedent myelodysplastic syndromes or myeloproliferative neoplasms, white blood cell count (

Background: Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. Vinca alkaloids such as vincristine (VCR), widely used in cancer treatment, are no exception, as their beneficial actions are often supplanted by resistance. In the present work we utilized HL-60 human leukemia cells and a VCR-resistant counterpart, HL-60/VCR cells, to determine potential interplay between SL metabolism and mitochondrial bioenergetics supportive of VCR resistance. Methods: Cellular SL alterations were assessed via a combination of enzyme immunoblot analysis and lipidomics. Mitochondrial function was evaluated in intact cells, permeabilized cells and isolated mitochondria using a comprehensive diagnostic assay system designed to directly interrogate oxidative phosphorylation (OXPHOS) kinetics. Informed by SL and bioenergetic readouts, experiments were designed to assess the therapeutic efficacy of co-targeting SL metabolism and mitochondrial flux to combat VCR resistance. Results: Relative to wild-type cells, HL-60/VCR presented with global alterations in SL composition, typified by upregulated expression of sphingosine kinase (SPHK1), which catalyzes formation of sphingosine 1-phosphate (S1P), glucosylceramide synthase (GCS), which catalyzes formation of glucosylceramides (GC), and acid ceramidase, responsible for ceramide hydrolysis. In support of these changes, VCR resistance was also characterized by increases in S1P, several molecular species of ceramide and GC, and changes in sphingomyelin (SM) molecular species. With respect to mitochondria, despite increased basal respiration in HL-60/VCR cells, direct interrogation of the mitochondrial network revealed intrinsic respiratory complex insufficiency, largely localized to complex I (CI). Importantly, forced ceramide accumulation in wild-type cells phenocopied the respiratory insufficiency observed in HL-60/VCR, and co-targeting SL metabolism and CI induced synergistic cytotoxicity in HL-60/VCR. Conclusions: Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against VCR resistance.

Key Points Distinct sphingolipid metabolism of AML with MDS-related changes defines unique sensitivity to nanoliposomal C6-ceramide. Vinblastine alters sphingolipid metabolism to enhance the sensitivity of AML to nanoliposomal C6-ceramide.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Retrospective studies have suggested that older adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL) have better survival rates when treated using a pediatric ALL regimen administered by pediatric treatment teams. To address the feasibility and efficacy of using a pediatric treatment regimen for AYA patients with newly diagnosed ALL administered by adult treatment teams, we performed a prospective study, CALGB 10403, with doses and schedule identical to those in the Children’s Oncology Group study AALL0232. From 2007 to 2012, 318 patients were enrolled; 295 were eligible and evaluable for response. Median age was 24 years (range, 17-39 years). Use of the pediatric regimen was safe; overall treatment-related mortality was 3%, and there were only 2 postremission deaths. Median event-free survival (EFS) was 78.1 months (95% confidence interval [CI], 41.8 to not reached), more than double the historical control of 30 months (95% CI, 22-38 months); 3-year EFS was 59% (95% CI, 54%-65%). Median overall survival (OS) was not reached. Estimated 3-year OS was 73% (95% CI, 68%-78%). Pretreatment risk factors associated with worse treatment outcomes included obesity and presence of the Philadelphia-like gene expression signature. Use of a pediatric regimen for AYAs with ALL up to age 40 years was feasible and effective, resulting in improved survival rates compared with historical controls. CALGB 10403 can be considered a new treatment standard upon which to build for improving survival for AYAs with ALL. This trial was registered at www.clinicaltrials.gov as #NCT00558519.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Immunotherapy targeting inhibitory pathways to unleash the antileukemia T-cell response is a promising strategy for the treatment of leukemia, but we must first understand the underlying molecular mechanisms. Eomesodermin (Eomes) and T-bet are both T-box transcription factors that regulate CD8+ T-cell responses in a context-specific manner. Here, we examined the role of these transcription factors in CD8+ T-cell immunity in AML patients. We report that the frequency of Eomes+T-betlow CD8+ T cells increased in newly diagnosed AML. This cell subset produced fewer cytokines and displayed reduced killing capacity, whereas depletion of Eomes by siRNA reversed these functional defects. Furthermore, Eomes bound the promoter of T-cell immunoglobulin and ITIM domain (TIGIT) and positively regulated the expression of this inhibitory receptor on patient-derived T cells. A high frequency of Eomes+T-betlow CD8+ T cells was associated with poor response to induction chemotherapy and shorter overall survival in AML patients. These findings have significant clinical implications as they not only identify a predictive and prognostic biomarker for AML, but they also provide an important target for effective leukemia therapeutics. Significance: These findings reveal that a high frequency of Eomes+T-betlow CD8+ T cells predicts poor clinical outcome in AML and that targeting Eomes may provide a therapeutic benefit against AML.

Patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) may benefit from salvage chemoimmunotherapy (CIT). To explore further the use of CIT in the pre-novel agent era, ECOG-ACRIN undertook a phase 2 trial (E2903) for R/R CLL utilizing pentostatin, cyclophosphamide, and rituximab (PCR) followed by a consolidation course of alemtuzumab. This trial enrolled 102 patients with a median age of 64 years. Treatment consisted of PCR for six cycles followed by alemtuzumab for either 4 or 12 weeks depending upon the initial response to PCR. The overall response after PCR (complete remission (CR), nodular partial remission (nPR), partial remission (PR)) was 55%. Major responses (CR, or nPR) were achieved in 6%. The median overall survival (OS) and the median progression- free survival (PFS) were 28 months and 12 months respectively. The most serious non-lethal adverse events were myelosuppression, febrile neutropenia, fatigue, nausea, and hyponatremia. PCR is an effective and well-tolerated nucleoside-based regimen for heavily pretreated CLL patients with R/R disease. The addition of alemtuzumab to CLL patients with a minor response (PR) or stable disease did not result in a significant number of higher responses (CR or nPR) nor an improvement in OS.

Acute myeloid leukemia (AML) represents a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors, and kinases that can cause epigenetic reshaping. It is unclear to what extent AML mutations drive chromatin 3D structure alteration and contribute to myeloid transformation. We first performed Hi-C and whole-genome sequencing in 25 AML patient samples and seven healthy donor samples, and identified recurrent alterations of A/B compartments, TADs, and chromatin loops that are unique to different subtypes. To investigate how altered chromatin organization contributes to transcriptional misregulation, we performed RNA-Seq, ATAC-Seq and CUT&ag for CTCF, H3K27ac, and H3K27me3 in the same AML samples. We identified extensive and recurrent AML-specific promoter-enhancer and promoter-repressor loops. We performed both CRISPR deletion and interference experiments and validated two repressor loops that downregulated cancer related genes IKZF2 and RTTN. Furthermore, by using our recently developed algorithm, we identified structural variation-induced enhancer-hijacking and repressor-hijacking events in AML samples. We further demonstrated the role of hijacked enhancers in AML cell growth by CRISPR screening, and the role of hijacked repressors by CRISPR de-repression. We performed whole-genome bisulfite sequencing in 20 AML and normal samples, and showed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Finally, by treating the AML cells with the DNA hypomethylating agent and performing triple knockdown of DNMT1/3A/3B, we demonstrated the impact of altered DNA methylation on gene expression and 3D genome organization. Overall this study provides an invaluable resource for leukemia studies and also highlighted the role of repressor-loops and hijacked cis-elements in gene regulation and human diseases. Citation Format: Jie Xu, Fan Song, Baozhen Zhang, Huijue Lyu, Mikoto Kobayashi, Ziyu Zhao, Ye Hou, Xiaotao Wang, Yu Luan, Bei Jia, Lena Stasiak, Qixuan Wang, Qi Jin, Qiushi Jin, Yihao Fu, Ross C. Hardison, Sinisa Dovat, Leonidas C. Platanias, Yue Yang, Tomoko Yamada, Aaron D. Viny, Ross L. Levine, David F. Claxton, James R. Broach, Hong Zheng, Feng Yue. Subtype-specific and structure variation induced 3D genome alteration in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2953.

Simple Summary Acute Myeloid Leukemia is an aggressive disease with poor outcomes. New targeted therapies that can boost the effects of currently used chemotherapy medications without added toxicity are needed. Targeting an overactive kinase, called the protein Kinase CK2 in AML, helps leukemia cells undergo cell death and helps certain chemotherapy drugs work better. Here, we present evidence that CX-4945, a CK2 inhibitor drug, effectively kills leukemia cells in mouse models and shows the mechanism of action responsible for these effects. Leukemia cells are more sensitive to a decrease in CK2 kinase levels than normal cells. Our results show that inhibiting CK2 kinase makes AML cells more susceptible to anthracycline-induced cell death. Anthracyclines like daunorubicin and doxorubicin are widely used to treat leukemia in children and adults. A rational combination of protein kinase CK2 inhibitors with the standard of care chemotherapy may help treat AML more effectively. Abstract 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.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML., Nucleophosmin (NPM1) gene mutation induces a specific gene expression program leading to acute myeloid leukaemia. Here, the authors show that mutant NPM1 activates a HOXB locus-associated long non-coding RNA which is essential for its associated oncogenic transcriptional program and leukaemia development.

Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (alloSCT) remains a major therapeutic challenge. While patients with longer remission after initial alloSCT are recommended to receive cell therapy (CT) such as 2ndalloSCT or donor lymphocyte infusion (DLI), survival for patients who relapse within 6 months of alloSCT has been dismal. We evaluated the outcomes of AML relapse after alloSCT to assess the impact of different treatments on long-term survival. One hundred and seventy-two patients with AML underwent alloSCT at the Penn State Cancer Institute from January 2014 to August 2019. Sixty-nine patients relapsed (median age, 60 years; range, 10–75). Of these, 4 patients underwent 2ndalloSCT, and 26 received DLI. One-year overall survival (OS) in all cases was 20.3% (95% CI: 11.8–30.4%). Patients with ECOG performance status (PS) 0–2 at relapse showed a better 1-year OS than those with PS 3–4. Median OS for patients who received chemotherapy only or chemotherapy with CT was 74 or 173.5 days, respectively (p

PURPOSE Impaired response to erythropoietin underlies ineffective erythropoiesis and anemia in myelodysplastic syndromes (MDS). We investigated whether treatment with lenalidomide (LEN), which augments erythropoietin receptor signaling in vitro, can restore and improve hemoglobin response to epoetin (EPO) alfa in patients with lower-risk, non-del(5q) MDS who have anemia that is refractory to or have low probability of benefit from treatment with recombinant erythropoietin. METHODS In a phase III, US intergroup trial, we randomly assigned patients to receive either LEN and EPO alfa or LEN alone following stratification by serum erythropoietin concentration and prior erythropoietin treatment. RESULTS A total of 195 evaluable patients were randomly assigned: 99 patients to the LEN-EPO alfa cohort and 96 to LEN alone. After four cycles of treatment, the primary end point of major erythroid response (MER) was significantly higher (28.3%) with the combination compared with LEN alone (11.5%) ( P = .004). Among 136 patients who completed 16 weeks of study treatment, 38.9% and 15.6% achieved MER, respectively ( P = .004). Additionally, minor erythroid response was achieved in 18.2% and 20.8% of patients, for an overall erythroid response rate of 46.5% versus 32.3%. Among LEN nonresponders, 38 crossed over to the addition of EPO alfa with 10 patients (26.3%) achieving a MER. Responses to the combined treatment were highly durable with a median MER duration of 23.8 months compared with 13 months with LEN alone. CONCLUSION LEN restores sensitivity to recombinant erythropoietin in growth factor–insensitive, lower-risk, non-del(5q) MDS, to yield a significantly higher rate and duration of MER compared with LEN alone (funded by the National Cancer Institute; E2905 ClinicalTrials.gov identifier: NCT02048813 ).