Primary Human Acute Lymphoblastic Leukemia Research Articles

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

CXCR4 mediates leukemic cell migration and survival in the testicular microenvironment.

The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B-cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12-CXCR4 as the leading signaling axis for B-ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient-derived xenograft (PDX)-ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2-like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4-mediated functions using an anti-CXCR4 antibody treatment completely abolished testicular infiltration of PDX-ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulation of Heterochromatin Landscape in T-Cell Acute Lymphoblastic Leukemia

Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute lymphoblastic leukemia (ALL). Deletion and/or loss of Ikaros function results in the development of high-risk leukemia. In the nucleus, Ikaros forms complexes with histone deacetylase complex, NuRD, and it participates in the formation of heterochromatin. The role of Ikaros-mediated formation of heterochromatin in tumor suppression in leukemia is unknown. We determined global genomic occupancy of Ikaros, global heterochromatin distribution, chromatin accessibility, DNA methylation landscape, and gene expression in primary human T-cell ALL (T-ALL), as well as in mouse T-ALL to analyze how Ikaros regulates heterochromatin landscape and gene expression in T-ALL. Results showed that Ikaros DNA occupancy is essential for the recruitment of histone deacetylase 1 (HDAC1), Polycomb repressive complex 2 (PRC2) and formation of facultative heterochromatin, as well as the formation of constitutive heterochromatin (characterized by H3K9me3 occupancy). T-ALL cells with deletion of both Ikzf1 alleles have severely impaired HDAC1 DNA occupancy and reduced H3K27me3. Re-introduction of Ikzf1 via retroviral transduction resulted in the restoration of H3K27me3 facultative heterochromatin, along with HDAC1 DNA occupancy. The H3K27me3 genomic distribution following Ikzf1 re-introduction showed high homology to the H3K27me3 genomic distribution in normal thymocytes. Analysis of H3K9me3 genomic distribution showed that Ikzf1 deletion results in dramatic redistribution of H3K9me3 global occupancy, with reduced H3K9me3 occupancy at pericentromeric loci. Reintroduction of Ikzf1 enhances H3K9me3 enrichment in pericentromeric loci, as well as at the promoters of genes that are involves in cellular proliferation. Analysis of DNA methylation distribution showed that Ikzf1 expression regulates global DNA methylation landscape. The presence of facultative heterochromatin, with enrichment of H3K27me3, inversely correlated with DNA methylation. Global analysis of chromatin accessibility revealed that Ikaros binding resulted in the loss of chromatin accessibility at over 3400 previously-accessible chromatin sites. Dynamic analyses demonstrate the long-lasting effects of Ikaros's DNA binding on heterochromatin distribution and chromatin accessibility. Analysis of gene expression in T-ALL with both Ikzf1 alleles and in Ikzf1-defficient cells (from Ikzf1-defficient T-ALL, and from Ikzf1-wild-type T-ALL following Ikzf1 deletion by CRISPR) showed that Ikaros-induced redistribution of facultative and constitutive heterochromatin results in the repression of several genes that are critical for cell cycle progression, PI3K-AKT-mTOR, and WNT signaling pathway. In conclusion, results suggest that Ikaros' tumor suppressor function in T-ALL occurs via global regulation of the heterochromatin, DNA methylation landscape, and chromatin accessibility, as well as via epigenetic regulation of transcription of the genes that play essential roles in signaling pathways that promote cellular proliferation. DisclosuresNo relevant conflicts of interest to declare.

Intrinsic Activation of the NLRP3 Inflammasome in Primary Human ALL Cells Is Regulated By Endogenous HMGB1 and Bone Marrow Macrophages

Inflammasomes are cytosolic signaling complexes that assemble in response to damage-associated molecular patterns (DAMPs). Intrinsic expression of the NLRP3 has been identified in acute lymphoblastic leukemia (ALL) cell lines and implicated in glucocorticoid resistance (Paugh W et al., 2015). The endogenous function of NLRP3 in ALL remains unknown. In this study, we use primary human ALL cells to demonstrate that NLRP3 is activated in primary ALL cells in response to chemotherapy stress, is regulated by the endogenously expressed “danger protein” HMGB1 and can be silenced through direct contact with bone marrow macrophages.To determine whether chemotherapy treatment of primary ALL cells induces NLRP3 expression, we treated CD19-selected cells from n=3 pediatric patients with ALL and lymphocytes from healthy donors with either 2nM doxorubicin or vehicle and compared NLRP3 expression by qPCR and Western blot at 24 hours. NLRP3 mRNA expression was increased by 4.5 + 0.8- fold over baseline in doxorubicin-treated ALL cells compared to no change in vehicle-treated ALL and undetectable levels in healthy lymphocytes. This corresponded to a 2-fold increase in NLRP3 protein expression was measured by Western Blot. Neither doxorubicin-treated nor vehicle-treated ALL expressed other NOD-family inflammasome proteins, including NLRP1 and NLRC4.In healthy monocytes, NLRP3 activates caspase-1 to induce transcription of inflammatory cytokines. To determine whether NLRP3 up-regulation has a similar function in primary ALL cells, we measured pro- and cleaved-caspase-1 in doxorubicin-treated primary ALL, vehicle-treated ALL and healthy lymphocytes. Primary ALL cells treated with doxorubicin expressed the transcriptionally active p20 subunit of caspase-1 by Western blot; vehicle-treated ALL cells and healthy lymphocytes did not. This corresponded to a 3.7 + 0.61-fold increase in global caspase-1 transcription by mRNA in doxorubicin-treated ALL cells and a significant increase in soluble IL1B production by ELISA (p<0.02). Treatment of primary ALL with staurosporin (an autophagy-inducer) and serum starvation up-regulated NLRP3 but not caspase 1/p20. Taken together, these data established proof-of-principle that primary ALL cells activate the NLRP3 inflammasome in response to clinically relevant cellular stress.The activating signal for NLRP3 assembly is unknown in leukemia. We have previously observed that DNA-binding DAMP high mobility group-box 1 (HMGB1) is endogenously expressed in ALL cells. To determine whether HMGB1 activates NLRP in leukemia, we knocked down HMGB1 by siRNA in primary ALL cells (n=3 patients) and measured NLRP3 and caspase-1/p20 at baseline and after treatment with doxorubicin vs. vehicle. HMGB1 siRNA ALL cells displayed at least a 2-fold decrease in NLRP3 protein expression 48 hours after knockdown compared to control siRNA cells that could not be rescued with doxorubicin treatment. Co-immunoprecipitation studies indicate that HMGB1 forms a binding complex in primary ALL cells.To determine whether NLRP3 expression in primary ALL is impacted by cells in the nonmalignant field, we co-cultured primary ALL cells with bone marrow-derived monocyte/macrophage, either directly or separated by 0.6 uM transwell. Unexpectedly, NLRP3 expression was completely abrogated in primary ALL cells when they were placed in direct, but not transwell, co-culture with macrophages. Direct co-culture of ALL with peripheral monocytes, however, did not abrogate NLRP3. These data indicate that direct interactions of ALL with macrophages in the nonmalignant bone marrow microenvironment have a negative regulatory effect on NLRP3 inflammasome activation.Taken together these data show, for the first time in primary pediatric ALL cells, that NLRP3 inflammasome is activated in response to chemotherapy cell stress that is regulated by endogenous HMGB1 expression and direct interactions with bone marrow macrophages. Studies are underway to define these interactions and their physiologic consequences for ALL cell survival in tne marrow niche. DisclosuresNo relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia Cells Bypass the Blood Brain Barrier to Enter the CNS Via a Neuronal Developmental Pathway

Background: In the absence of prophylactic treatment, CNS disease involvement will occur in 30-70% of acute lymphoblastic leukemia (ALL) patients. CNS metastasis is observed in all ALL subtypes, suggesting that lymphoblasts utilize a conserved mechanism to invade the CNS. CNS ALL relapse predicts a poor outcome, but treatment options remain limited due to a minimal understanding of the biologic processes involved.PI3K regulates in many cell activities, including proliferation, migration, and apoptosis. The δ isoform of PI3K is preferentially expressed in immune cells. The PI3Kδ inhibitor, idelalisib, is approved for use in combination with rituximab for treatment of chronic lymphocytic leukemia. Previously, the PI3Kd tool compound, GS-649443 (443), which has more favorable murine pharmacokinetic properties, was evaluated in in vivo models of acute lymphoblastic leukemia (Yao H, et al. ASH meeting, 2016). Here we report on the mechanisms of ALL CNS invasion and the potential for PI3Kd inhibition to block CNS disease.Results: Our prior study of 443 in Nalm-6 (N6) B-ALL mice demonstrated that PI3Kd inhibition prevents ALL CNS disease progression. All control mice showed symptoms of CNS involvement including hind limb paralysis, but only 17% of 443-treated mice developed paralysis at their clinical endpoint. There was no difference in ALL burden or apoptotic rate in bone marrow (BM) or spleen of treated vs. control mice at matched time points. In contrast, cerebrospinal fluid (CSF) blast counts were significantly decreased in 443-treated mice.To confirm our results were not isolated to the N6 model, we tested the efficacy of PI3Kδ inhibition in mice engrafted with primary human ALL (hALL). Treated mice demonstrated a modest decrease in peripheral disease burden. Similar to the results in N6 mice, 443 markedly decreased CNS disease burden in the hALL model (Fig. 1).Examination of 443 pharmacokinetics as well as mitotic and apoptotic indices of CSF blasts isolated from 443 vs. vehicle-treated mice indicated that PI3Kd inhibition did not directly impact growth of ALL in the CNS. We therefore investigated whether 443 prevented migration of blasts from the periphery into the CNS. We found that 443 decreased Transwell® (TW) migration of N6 and hALL cells toward SDF-1 or human CSF. We next interrogated the signaling pathways downstream of PI3K that are implicated in cell motility and found that 443 suppressed the phosphorylation of myosin light chain 2 in ALL cells. To identify candidate regulatory molecules upstream of this pathway, we performed a microarray analysis of N6 cells isolated from GS-443 vs. vehicle-treated mice. This showed that multiple genes within focal adhesion and contractility pathways were downregulated in vivo in 443 treated blasts, including integrin α6.α6, a subunit of cell surface laminin receptors, is expressed in the majority of ALL cases. Laminin deposition is found in the extracellular matrix (ECM) of CNS microvessels and meninges, where it coordinates α6-dependent neuronal progenitor cell pathfinding. Using confocal microscopy, we determined that N6 and hALL cells in circulation are unable to breach the blood brain barrier. Rather, we identified that ALL cells migrate into the CNS along vessels that pass directly between vertebral or calvarial BM and the subarachnoid space, and that the ECM of these bridging vessels is enriched in laminin (Fig. 2). In in vitro invasion assays, we confirmed that ALL migration to CSF is enhanced by laminin in an α6-dependent manner. Lastly, the in vivo effects of α6 were investigated in the N6 model. Mice were treated with a6 neutralizing antibodies beginning on post-engraftment day 1 through the development of clinical symptoms requiring sacrifice. Anti-α6-treated mice showed cachexia at their clinical endpoint but none had severe CNS symptoms, whereas 100% of isotype control-treated mice showed hind limb paralysis. No difference in ALL burden or apoptotic rate was observed in the BM or spleen of anti-α6 vs. isotype control groups. In contrast, there was a significant decrease in the number of blasts harvested from the CSF of anti-α6-treated mice (Fig. 3).Conclusion: Our results identify a novel ALL metastasis mechanism. Expression of integrin α6, common in ALL, allows cells to co-opt neural migratory pathways to invade the CNS. The potential for PI3Kδ inhibition to block development of CNS ALL warrants further investigation in a clinical study. [Display omitted] DisclosuresYao:Gilead Sciences, Inc.: Research Funding. Warner:Gilead Sciences, Inc.: Research Funding. Price:Gilead Sciences, Inc.: Research Funding. Olivere:Gilead Sciences, Inc.: Research Funding. Cantelli:Gilead Sciences, Inc.: Research Funding. Ridge:Gilead Sciences, Inc.: Research Funding. Ngo:Gilead Sciences, Inc.: Research Funding. Ravichandran:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences, Inc.: Employment. Sipkins:Gilead Sciences, Inc.: Research Funding.

Repurposing anthelmintic agents to eradicate resistant leukemia

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia.

Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.

ARID5B Influences Antimetabolite Drug Sensitivity and Prognosis of Acute Lymphoblastic Leukemia.

Treatment outcomes for childhood acute lymphoblastic leukemia (ALL) have improved steadily, but a significant proportion of patients still experience relapse due to drug resistance, which is partly explained by inherited and/or somatic genetic alternations. Recently, we and others have identified genetic variants in the ARID5B gene associated with susceptibility to ALL and also with relapse. In this study, we sought to characterize the molecular pathway by which ARID5B affects antileukemic drug response in patients with ALL. We analyzed association of ARID5B expression in primary human ALL blasts with molecular subtypes and treatment outcome. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events. ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs (i.e., 6-mercaptopurine and methotrexate), without significantly affecting sensitivity to other antileukemic agents. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest. At the molecular level, the cell-cycle checkpoint regulator p21 (encoded by CDKN1A) was most consistently modulated by ARID5B, plausibly as its direct transcription regulation target. Our data indicate that ARID5B is an important molecular determinant of antimetabolite drug sensitivity in ALL, in part, through p21-mediated effects on cell-cycle progression.

Abstract 871: Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 871.

Abstract A81: Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia

Abstract The ability of Toll-like receptor (TLR)-induced innate immune responses to activate adaptive immune responses offers considerable therapeutic potential for cancer treatment, most likely as part of combination therapies. The potent immunostimulatory capacity of TLR agonists provides means to generate antigen-specific antitumor T cell responses from antigen-nonspecific innate immune stimulation. B cell precursor (BCP) acute lymphoblastic leukemia (ALL) exhibits the very low mutational burden common to other pediatric cancers. Despite this limited neo-antigenicity, we previously reported the ability of CpG oligodioxynucleotides (ODN), a ligand for TLR9, to mediate anti-leukemia activities that establish long-term protection against ALL outgrowth in a transplantable syngeneic model using cell lines. CpG ODN also reduced the burden of primary human ALL in mouse xenografts, primarily via induction of NK cell-mediated cytotoxicity. However, our recent demonstration that endosomal TLR agonists generated distinct effects on pre-leukemic BCP cells, coupled with the divergent effects of TLR ligands on the immunogenicity of primary human ALL cells suggest that there are significant variables that contribute to the overall outcome of TLR stimulation in the context of BCP cell malignancy. In this study, we compared the ligands for TLR3 (polyI:C), TLR7/8(R848) and TLR9 (CpG ODN) for their ability to elicit control of primary ALL cells and correlated protection with early innate immune activation to identify the necessary component of a long-term protective anti-leukemic response. We adoptively transferred syngeneic primary ALL cells to wild-type mice then administered TLR ligands starting at day 7 for 3 doses every 4 days. 21 days after leukemia challenge, we evaluated the disease burden in the peripheral blood, spleen, and bone marrow of recipients. While a significantly lower disease burden was observed in all three anatomical compartments of mice treated with polyI:C, CpG ODN- and R848-treated mice failed to control the early outgrowth of transferred cells in the spleen and bone marrow, respectively. While R848-treated mice had a slightly delayed disease progression with median survival (MS) of 40 days compared to that of 28 days of PBS-treated control group, CpG ODN treatment conferred a significant survival advantage where over 57% of treated mice achieved a prolonged disease-free status. Despite successfully inducing systemic anti-ALL responses, poly I:C treatment failed to confer durable protection, achieving only a modest increase in disease-free survival in treated mice (MS=42 days). Moreover, CpG ODN-induced long-term survival in the absence of effective early immune-mediated control of ALL in spleen implies that bone marrow is not only an important homing niche supporting the survival and proliferation of leukemia blasts prior to migration into the circulation, but a critical target site for controlling disease progression. Furthermore, to investigate the early innate immune responses associated with the differential overall effects induced by TLR agonists, we evaluated the activation status of innate immune system using RAG-knockout mice. A single administration of CpG ODN, but not polyI:C nor R848, was sufficient to rapidly induce anti-ALL immune responses involving activated natural killer and antigen-presenting cells in the bone marrow of the mice bearing ALL in 4 days. This suggests that CpG ODN has superior capacity to initiate innate immune-mediated anti-ALL immune responses and activate subsequent antigen-specific T cell responses. TLR-mediate innate immune responses induced early in life are distinct from that induced in adult life. Given the peak incidence of pediatric ALL between 3-5 years of age, we are currently examining the influence of age on the TLR-induced anti-ALL immune responses to further evaluate the therapeutic benefits of TLR ligands. Our results demonstrate the differential magnitude of tissue-specific innate immune-mediated control of leukemia outgrowth induced by TLR stimulations correlating with durable disease-free survival. These findings provide mechanistic explanation for TLR-mediated protective immune responses and highlight the potential target site necessary for effective elimination of leukemia cells. Citation Format: Sumin Jo, Peter van den Elzen, Gregor S.D Reid. Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A81.

Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.

Genome-wide DNA replication timing (RT) profiles reflect the global three-dimensional chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of cellular epigenetic state. Thus, normal differentiation involves reproducible changes in RT, and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome invitro. In contrast, the ability of many human ALL cell populations to expand when transplanted into highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for bromodeoxyuridine incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistently strong retention in xenografts of the original patient-specific RT features. Moreover, in a case in which genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results indicate that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in ALL.

PI3Kδ Inhibition Suppresses Central Nervous System Involvement of Acute Lymphoblastic Leukemia

The majority of children and adults who experience relapse of acute lymphoblastic leukemia (ALL) will die from the disease. The presence of leukemic blasts within the cerebrospinal fluid (CSF) is an important predictor of disease recurrence in both the bone marrow (BM) and central nervous system (CNS), a frequent site of disease involvement. In the absence of intrathecal chemotherapy or craniospinal irradiation, 30%–50% of patients will in fact develop CNS disease. All patients therefore receive CNS prophylaxis. High-risk patients require intensive CNS-directed treatment that causes toxic side effects and can impact cognitive development in children. Patients who develop recurrence in the CNS have limited treatment options and an extremely poor prognosis.The enzyme PI3K plays an important role in many biological effects including cell proliferation, migration, and apoptosis. The d isoform of PI3K is specifically expressed in immune cells. The PI3Kd inhibitor idelalisib is approved for use in combination with rituximab for the treatment of chronic lymphocytic leukemia (CLL). However, little is known about the efficacy of PI3Kd inhibition in ALL. Here, the PI3Kd inhibitor GS-649443, a potent and specific in vivo tool compound, was evaluated in a xenograft mouse model.The in vivo effects of GS-649443 were investigated in a Nalm-6 pre-B ALL SCID model. At approximately 40 days postengraftment, untreated mice all developed symptoms of CNS involvement and displayed hind limb paralysis. Hind limb paralysis is the clinical endpoint for sacrifice, occurring prior to death from progressive BM disease. Mice were treated with GS-649443 by oral gavage beginning on postengraftment day 1 and continuing until the development of clinical symptoms requiring sacrifice (hind limb paralysis, weight loss >20%, respiratory or other distress). Only 17% of GS-649443–treated mice developed hind limb paralysis at their clinical endpoint, while 100% of vehicle-treated mice showed hind limb paralysis. In addition, mice treated with GS-649443 or vehicle control were paired. When either mouse in a treatment pair reached a clinical endpoint, both were sacrificed so disease burden in individual organs could be compared at matched time points. There was no difference in tumor burden or leukemic cell apoptotic rate in the BM of treated vs vehicle control groups. In contrast, there was a significant decrease in the number of leukemic blasts harvested from the CSF of treated mice (Fig. 1). Consistent with the inhibition of CNS disease progression, GS-649443 significantly prolonged the survival of treated mice (Fig. 2).While compromised blood-brain barrier in leukemic mice may allow therapeutic targeting of CNS disease, it is unknown whether GS-649443 enters the CNS in healthy rodents. Given the well-described effects of idelalisib on CLL cell migration, we hypothesized that PI3Kd blockade impacts disease in this ALL model by impairing leukemia migration into the CNS. To investigate the in vitro effect of GS-649443 on ALL cell migration, transwell migration assays with SDF-1 as chemoattractant were performed. Both GS-649443 and idelalisib suppressed migration of Nalm-6 and primary human ALL cells toward SDF-1.Lastly, the effects of GS-649443 in combination with conventional chemotherapy were examined. GS-649443 alone did not cause significant cytotoxicity of Nalm-6 in vitro, however, treatment with GS-649443 after cytarabine significantly increased apoptosis. Leukemic mice (20 days post-engraftment) were then treated with cytarabine (days 1–5) in combination with GS-649443 or cytarabine alone (days 1–7) for 1 cycle. In contrast to the single-agent study, significant decreases in the number of leukemic blasts harvested from both the BM and CSF of treated mice were observed, suggesting that PI3Kd inhibition sensitized ALL cells to cytarabine chemotherapy (Fig. 3). This decrease in residual disease after combination therapy led to significantly prolonged survival in GS-649443–treated mice (Fig. 4).Taken together, this study provides evidence that PI3Kd inhibition prevents ALL progression in the CNS. In addition, GS-649443 and conventional chemotherapy appear to synergize to decrease residual BM disease. The potential for PI3Kd inhibition to improve chemotherapy response and impede development of CNS disease in ALL warrants further investigation in a translational clinical study. [Display omitted] DisclosuresYao:Gilead Sciences, Inc.: Research Funding. Price:Gilead Sciences, Inc.: Research Funding. Olivere:Gilead Sciences, Inc.: Research Funding. Warner:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences: Employment. Sipkins:Gilead Sciences, Inc.: Research Funding.

Transcriptional Repression of the LMO2 Oncogene By Ikaros in T-Cell Acute Lymphoblastic Leukemia

LIM domain only protein 2 (LMO2) is a regulator of hematopoiesis and an oncogene that is overexpressed in a subset of T-cell acute lymphoblastic leukemia (T-ALL). Overexpression of LMO2 in T-ALL is associated with a poor prognosis. The mechanisms that regulate LMO2 expression in T-ALL are still unknown. Here, we present evidence that expression of LMO2 in T-ALL is regulated at the transcriptional level by Ikaros, a tumor suppressor protein whose deletion is associated with the development T-ALL. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in primary human acute lymphoblastic leukemia cells and in cell lines demonstrated Ikaros occupancy of the LMO2 promoter. Ikaros binding at the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL and B-ALL cells. The role of Ikaros in the regulation of LMO2 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with siRNA resulted in increased transcription of LMO2 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strongly reduced transcription of LMO2. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of LMO2. Transduction of these cells with Ikaros-containing retrovirus, results in a sharp reduction of LMO2 expression. Since Ikaros function in T-ALL is negatively regulated by the pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition can enhance Ikaros-mediated transcriptional repression of LMO2. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor, resulted in reduced expression of LMO2 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the LMO2 promoter. Ikaros knock-down restored high expression of LMO2 in T-ALL cells that were treated with CK2 inhibitors. These data show that Ikaros is a major regulator of LMO2 transcription in T-ALL and that CK2 inhibition requires Ikaros activity to repress LMO2 transcription. Increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at the LMO2 promoter suggesting that Ikaros regulates LMO2 transcription via chromatin remodeling. In conclusion, these results provide evidence that expression of the LMO2 oncogene is regulated by Ikaros and CK2 in T-ALL. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in a preclinical model of T-ALL. The presented data reveal a novel mechanism of therapeutic action for CK2 inhibitors - repression of LMO2 expression via Ikaros. These results provide a rationale for the use of CK2 inhibitors in T-ALL with LMO2 overexpression. DisclosuresNo relevant conflicts of interest to declare.

Targeting of a novel signaling program in Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL)

Abstract Molecular crosstalk between the interleukin-7 receptor (IL-7R) and the pre-B cell receptor (pre-BCR) represents a key developmental checkpoint regulating early B cell proliferation and differentiation. Dysregulation of these pathways has been linked to early lymphoid malignancies. While Philadelphia chromosome-like acute lymphoblastic leukemias (Ph-like ALL) lack the canonical chromosome translocation present in BCR-ABL1+ (Ph+) ALL, these tumors share a similar gene expression profile. Notably, however, Ph-like ALL exhibits a poor clinical prognosis and is refractory to most available therapies. A subset of Ph-like ALL results from alterations in the IL-7R signaling pathway, with gain-of-function mutations in IL-7Rα and loss-of-function mutations in the adaptor, SH2B3, in numerous samples. In this study we directly assess the signaling program downstream of the IL-7R and SH2B3 and its role in disease pathogenesis. Primary human Ph-like ALL tumors display constitutive STAT5 activation, a phenotype recapitulated by in vitro analysis of transduced cell lines expressing an activating IL-7Rα mutation or loss of SH2B3. Our results suggest that both IL-7Rα and SH2B3 mutations can individually contribute to this altered signaling program and may be capable of inducing oncogenic transformation. We have also developed a mouse model that allows for inducible expression of an activating IL-7Rα mutation for future in vivo analysis. Current work is focused on using mass spectrometry and phosphoproteomics to determine downstream signaling aberrations in both primary tumors and our stably transduced cell lines. The results from these studies may lead to new therapeutic targets for high-risk ALL patients.

Alarmin High Mobility Group Box 1 (HMGB1) Activates Alternative NFkB Signaling in Bone Marrow Macrophages in the Leukemia Microenvironment

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment.We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models.To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1.Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion.These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

A Phase I Study of Carfilzomib for Relapsed or Refractory Acute Myeloid and Acute Lymphoblastic Leukemia

Background: The outcome for patients with relapsed or refractory acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) remains extremely poor; new chemotherapy agents with activity against these leukemias are urgently needed. Proteasome inhibition is a promising therapeutic target in acute leukemias with established efficacy in several other hematologic malignancies. Several published studies have demonstrated the potent in vitro anti-leukemic activity of the selective proteasome inhibitor carfilzomib (CFZ) in leukemia cell lines as well as in primary human AML and ALL blasts. Given the favorable toxicity profile of CFZ, there was a strong rationale to begin the clinical study of this agent in the treatment of acute leukemias. We performed a phase I, single arm, single center, open label study of CFZ in adult patients with relapsed or refractory AML and ALL.Objectives: The primary objective was to determine the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of CFZ in adult patients with relapsed/refractory AML and ALL. Secondary objectives included: determination of disease response, time to response, remission duration, progression-free survival, event-free survival, and overall survival. The study was designed to determine the safety and tolerability of carfilzomib in this patient population.Patients/Methods: Patients with relapsed or refractory AML or ALL after ³ 1 line of therapy, with good performance status and organ function were eligible. CFZ was given on days 1, 2, 8, 9, 15, 16, 22, and 23 in a 28-day cycle at escalating doses of 36, 45, and 56mg/m2. All dose levels included lead-in doses on days 1, 2, 8 and 9. Up to 6 cycles of treatment were allowed.Dose escalation was performed using a 3+3 design; MTD was defined as the highest dose level where DLT occurred in less than 1 of 3 or 2 of 6 patients. Disease response was determined using International Working Group (IWG) criteria for AML.Results: 18 patients were enrolled from Dec 2010-July 2014. Eight patients discontinued prior to completion of cycle 1 for reasons other than study-related toxicity and were replaced.The median age was 70 years (range 32-78). 12 of the 18 patients were male. 17 patients had AML while 1 patient had ALL. The median number of prior therapies was 2 (range 1-4). 2 patients had undergone prior allogeneic stem cell transplantation. The median time from diagnosis to start of protocol was 9.7 months (range 1.4-102.0).Median white blood cell (WBC) count at study start was 3.2x109/L (range 0.6-42.2), with median peripheral blood blasts of 18% (range 0-62), and bone marrow blasts 45.5% (range 11-92). 7 of the 17 patients with AML had complex cytogenetics.10 of the 18 patients were evaluable for disease response. Of the 10 evaluable patients, 2 patients achieved a partial response (PR), and 4 additional patients had stable disease (SD). The median duration on study was 21.5 days (range 1-64), with median overall survival (OS) of 1.9 months.CFZ was generally well tolerated; no DLTs were observed in any cohort. The most common grade 3/4 non-hematologic toxicities regardless of attribution were: dyspnea (17%), activated partial thromboplastin time prolongation (17%), intracranial hemorrhage or hematoma (11%), infection (11%), CHF exacerbation (11%), and hypophosphatemia (11%).Conclusions: Administration of CFZ was well tolerated, and a MTD was not established. The recommended dosage for further studies is 56mg/m2. In this limited phase 1 study of heavily pre-treated patients, we observed modest anti-leukemic activity. However, additional clinical trials incorporating CFZ in combination with other chemotherapeutic agents are now needed to assess the potential efficacy of CFZ in the treatment of AML and ALL.Abstract 5292. Table 1IDDose LevelDiagnosis# of Prior Lines of TherapyPrior SCT# of CyclesBest Response11 (36mg/m2)AML1N3SD21 (36mg/m2)AML3Y<1NA31 (36mg/m2)AML2N2PR41 (36mg/m2)AML2N2SD52 (45mg/m2)AML2N1PD62 (45mg/m2)AML2N<1NA72 (45mg/m2)AML3N<1NA82 (45mg/m2)AML2N<1NA92 (45mg/m2)AML2N2SD102 (45mg/m2)AML3N<1NA112 (45mg/m2)AML2N2SD122 (45mg/m2)AML2N1PD133 (56mg/m2)AML3N1PD143 (56mg/m2)AML4Y2PR153 (56mg/m2)AML1N1PD163 (56mg/m2)AML3N<1NA173 (56mg/m2)ALL1N<1NA183 (56mg/m2)AML2N<1NAAbbreviations: NA- Not Applicable; SD- Stable Disease; PR- Partial Response; PD- Progressive Disease DisclosuresOff Label Use: We performed a phase I, single arm, single center, open label study of the selective proteasome inhibitor carfilzomib (CFZ) in adult patients with relapsed or refractory AML and ALL. CFZ is approved for the treatment of patients with multiple myeloma who have already received at least two other treatments including bortezomib and an immunomodulatory agent (lenalidomide and/or thalidomide), and whose disease has progressed (got worse) on their last therapy or within 60 days of their last therapy.. Vij:Onyx Pharmaceuticals, Inc.: Honoraria, Research Funding.

A Novel Platform For The Identification Of Stromal-Derived Factors That Enhance Acute Lymphoblastic Leukemia Cell Survival In The Context Of Maintenance Chemotherapy

IntroductionChildhood B-lineage acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. ALL is highly responsive to intensive, multi-agent chemotherapy, and most children enter remission; 25% of children relapse. Furthermore, cure requires an additional two years of low intensity maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. It is known that serum levels of maintenance chemotherapy drugs are not highly cytotoxic for ALL cells, and it is thought that bone marrow stromal cells (BM-SC) provide trophic signals to leukemia cells that result in increased resistance to standard therapy. If anti-apoptotic signals from stromal cells to leukemia cells were known, new molecular targets for leukemia therapy could be developed. We are developing a conceptually simple screening system that will identify stromal cell-derived molecules that support ALL cell survival in the setting of 6MP and methotrexate. Our hypothesis is based on the observation that in serum-free conditions, BMSC prevent apoptosis of primary ALL cells. We reason that if we interfere with the production of key BMSC-derived trophic factors, an increase in ALL cell apoptosis in vitro will be seen. MethodsThe system has 3 components: (1) human marrow stromal cells; (2) primary ALL cells (not established cell lines); and (3) siRNAs to knockdown candidate stromal genes. We employ a human mesenchymal stromal cell (MSC) line immortalized with a human TERT gene that has been shown to be representative of primary human stroma. 20,000 BMSC are placed in wells of a 96-well plate and treated with siRNA. 48 hours later, 30,000 primary human ALL cells are added in serum-free media. After 5 days, viable ALL cells are measured by flow cytometry. Results(1) The addition of pharmacological concentrations of 6-MP to BMSC/ALL co-cultures does not significantly increase primary ALL apoptosis. Co-cultures were established as above and 6-MP was added at 1 micromolar. ALL survival was measured at 5 days. No increase in ALL survival was seen in the 6-MP containing cultures. On unmanipulated stroma cultured without 6-MP, 1948.8 (± 397.0) viable ALL cells were recovered; on manipulated stroma in the presence of 6-MP, 1698.5 (±299.3) (p value = 0.12). (2) Interference with stromal cell protein synthesis significantly increases ALL cell apoptosis. We hypothesize that interference with new protein synthesis might reduce stromal support of ALL cells. To test this we treated BMSC for 6 hours with 25 micrograms of G418 which irreversibly blocks polypeptide synthesis. Wells were then washed with serum-free medium. Stromal cells remained viable for up to 1 week. However, ALL cells apoptosis was much higher on such treated stromal cells (32.7±18.5 viable ALL cells on G418-treated stroma versus 1948.8 ± 397.0 on unmanipulated stroma, p value< 0.001)(Figure 1). (3) Interference with global stromal cell RNA transcription significantly increased ALL cell apoptosis. Triptolide is a molecule that irreversibly inhibits RNA transcription. BMSC were treated for 6 hr with 1 microgram triptolide and washed with serum-free medium. Stromal cells remain viable for up to 1 week. ALL cell apoptosis was significantly reduced by triptolide treatment of stroma (not shown in Figure 1). The number of viable ALL cells recovered from triptolide-treated stroma was 252.3±113.2, compared to 996.6±239.2 on unmanipulated stroma (p< 0.001). (4) Knockdown of CXCL12 gene expression in stromal cells signficantly increase ALL cell apoptosis. For proof of principle, we have started with CXCL12, a gene shown in CLL to contribute to leukemia cell survival. BMSC were treated with 0.3 picomolar anti-CXCL12 siRNA for 24 hr. As measured by quantitative RT-PCR, CXCL12 gene transcription was reduced by 80%. Moreover, there was a 53.5% reduction in ALL cell viability in ALL cells co-cultured with CXCL12 siRNA-treated stroma compared to ALL cells co-cultured with untreated stroma (p value< 0.001) (Figure 1). [Display omitted] ConclusionsThis BMSC cell/primary ALL cell system can be used to identify stromal cell genes that prevent apoptosis of primary ALL cells. The simple system can be scaled up to allow high throughput screening of candidate genes that have antagonistic or additive effects to maintenance chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Identification Of BCL6 As a Therapeutic Target In MLL-Rearranged ALL

BackgroundBCL6 has been identified in diffuse large B cell lymphoma (DLBCL), where it is frequently translocated to immunoglobulin loci and acts as a protoncogene and transcriptional repressor. We found in Ph+ acute lymphoblastic leukemia (ALL) that BCL6 is involved in a novel form of drug resistance to tyrosine kinase inhibitors (TKI) by protecting cells from p53-mediated apoptosis (Duy et al., Nature 2011). Our current study is focusing on the function of BCL6 in Non-Ph+ALL based on the finding that high BCL6 levels represent a predictor of poor clinical outcome. ResultsWe analyzed the gene expression data from 207 children with high-risk B cell precursor ALL enrolled in the pediatric clinical trial (COG P9906) and found that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (OS p=0.007; RFS p=0.02). Furthermore, multivariate analyses showed that patients with high BCL6 expression levels and established predictors of poor clinical outcome such as high white blood counts (WBC), a positive minimal residual disease (MRD) or mutations in the tumor suppressor IKZF1 had a significantly poor OS and RFS. Matched sample pairs from 49 patients at diagnosis and relapse showed that BCL6 levels were increased at relapse compared to diagnosis (p=0.003). We next studied BCL6 protein levels in Non-Ph+ cell lines and childhood ALL patient samples and found that BCL6 levels are particularly high in MLL-AF4 ALL (n=19). Surprisingly, the patients from the clinical trial that had high BCL6 levels and MLLR rearrangements were those with the worst clinical outcome (OS p= 0.0009 and RFS p= 0.000208). We next tested if the MLL-AF4 oncogene drives aberrant BCL6 expression. First, we performed a ChIP-analysis with the oncoprotein MLL-AF4 and found that the BCL6 promoter is directly bound by MLL-AF4, suggesting that MLL-AF4 may indeed drive BCL6 expression. We then performed a BCL6 Western Blot of inducible MLL-AF4-transgenic pro-B cells, demonstrating that activation of the MLL-AF4 transgene is sufficient to induce ∼10-fold upregulation of BCL6 protein levels. We conclude that aberrant expression of BCL6 in childhood ALL can be the direct consequence of MLL-AF4 activity. To further elucidate the BCL6 signaling pathway, we performed a ChIP-analysis with human MLL-AF4 leukemia cells and found that tumor suppressor genes (e.g. CDKN1B and BACH2) and B cell linage specific genes like PAX5 and CD19 are repressed by BCL6, potentially explaining the mixed lineage phenotype of MLLR driven B-ALL. We used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6-/- mice to decipher the function of BCL6. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transduced B cell progenitor cells from BCL6-/- and BCL6+/+ mice with Nras using an inducible oncogenic TET-NRASG12D system. Similar to MLL-AF4 driven cells, activation of NRASG12D results in higher BCL6 protein expression. Strikingly, BCL6-deficiency results in a failure of NRASG12DALL cells to initiate leukemia. Clinical relevanceTo verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human childhood ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for children with MLL-AF4 leukemia, we treated human MLL-AF4 driven primary human xenograft cells with a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI). Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. ConclusionsThese findings identify BCL6 as an important factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat childhood ALL. Aberrant expression of BCL6 in MLL-AF4 ALL is the direct consequence of MLL-AF4 activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors (e.g. RI-BPI) with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures:No relevant conflicts of interest to declare.

Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling

AbstractThe efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line– and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.

Abstract 4684: Vitamin D may impair glucocorticoid cytotoxicity in acute lymphoblastic leukemia cells

Abstract Vitamin D deficiency has been associated with increased incidence and mortality in several types of cancer. Vitamin D has been shown to be cytotoxic to cancer cells in vitro, and may act synergistically with chemotherapy to kill cancer cells. Although vitamin D deficiency is common in children with cancer, the clinical implications of this remain unclear. Since acute lymphoblastic leukemia (ALL) is the most common type of cancer in children and relapsed leukemia remains a major problem, the present study was designed to test our hypothesis that active Vitamin D (Calcitriol) would induce apoptosis in ALL cells and potentiate the effect of chemotherapy on ALL cells. We cultured several human ALL cell lines and primary human ALL cells with and without Calcitriol, and quantified survival using trypan blue exclusion. Contrary to our hypothesis, Calcitriol did not negatively affect the viability or impair proliferation in the human ALL cell lines RS4; 11, SD1, BV173, or RCH-ACV, or in primary human ALL cells US7 or TXL 2. We then tested whether Calcitriol would synergize with dexamethasone, a glucocorticoid used almost universally in the treatment of childhood ALL. Again, contrary to our hypothesis, we found that rather than augment dexamethasone-induced cytotoxicity, supraphysiological doses of Calcitriol protected RS4;11 cells from the cytotoxic effect of 72 hour exposure to 100 nM dexamethasone (dexamethasone alone: 10±5% of initially plated cells viable; dexamethasone plus 300 nM Calcitriol: 27±14% of initially plated cells viable, n=4, p=0.04). More physiological doses of Calcitriol (10nM) showed similar protection (12.5±5% vs 31.5±19% of initially plated cells were viable at 72 hrs, n=4, p=0.05). Preliminary flow cytometry using Annexin/7AAD suggests that Calcitriol decreases dexamethasone-induced apoptosis in RS4;11 cells (dexamethasone alone: 94% of cells in apoptosis; dexamethasone plus 100 nM Calcitriol: 75% of cells in apoptosis, n=1) In summary, the active form of vitamin D appears to protect ALL cells from dexamethasone. This finding could be important, since glucocorticoid sensitivity of ALL cells is highly predictive of outcome and likelihood of relapse. Our findings could have major health implications given the high prevalence of Vitamin D deficiency in children and adolescents worldwide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4684. doi:10.1158/1538-7445.AM2011-4684