Infant Acute Lymphoblastic Leukemia Patients Research Articles

Targeted Inhibition of the MLL Transcriptional Complex By Proteosome Inhibitors Elicits a High Response Rate in Relapsed/Refractory MLL Rearranged Leukemia

Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients.To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (NSG) mice. All samples engrafted and expanded in NSG mice, leading to overt leukemia with a latency of 49 to 276 days. Purification of leukemic blasts from a single moribund mouse yielded on average 108 cells, providing sufficient material to screen large numbers of compounds. In vitro conditions were defined that support growth in 40% of the patient specimens, allowing for a more accurate determination of drug sensitivity. Growth in vitro was associated with early onset of disease in NSG xenografts and younger age at presentation, allowing us to evaluate patient samples that represent aggressive high risk disease. Using this system, we tested bortezomib in addition to 28 other drugs, including standard ALL therapeutic agents as well as targeted kinase inhibitors and inhibitors of epigenetic marks. Three classes of agents were active in this system: anthracyclines, histone deacetylase inhibitors (HDACi), and the proteasome inhibitor bortezomib. In contrast to anthracyclines and HDACi, where IC50 values were on par with those reported in the literature for primary childhood ALL samples, MLLr infant samples required 10-100 fold less bortezomib to induce toxicity.Bortezomib has been shown to mediate responses through several mechanisms, including NFKB inhibition, stabilization of cell cycle regulatory proteins, and induction of apoptosis. Recently, proteasome inhibition has been demonstrated to lead to accumulated MLL fusion protein levels, triggering apoptosis and cell cycle arrest in MLLr cell lines. To determine if NFKB inhibition also plays a role, we evaluated cellular concentrations of the activated NFKB transcription factor, but failed to see decreased levels when MLLr cells were treated with bortezomib. Bortezomib has also been shown to deregulate ubiquitin stores and deplete histone H2B ubiquitination (H2Bub), an epigenetic mark that is linked to histone methylation and expression. Recently, several groups have demonstrated that H2Bub is required for DOT1L activity and HOX gene expression. We therefore evaluated H2Bub levels in bortezomib-treated patient samples and confirmed depletion of this epigenetic mark. Furthermore, patient samples treated with bortezomib downregulated both the MLL gene expression signature and signatures of downstream targets, such as cMYC, demonstrating that the MLL transcriptional program is inhibited in the presence of bortezomib. ChIP-seq is underway to map H2Bub and H3K79 methylation changes genome wide in response to treatment with bortezomib.The HDACi vorinostat and bortezomib have both been evaluated in Phase I and II pediatric leukemia clinical trials. Based on the safety and efficacy from these earlier studies, we treated 6 relapsed/refractory MLLr leukemia patients with a chemotherapy regimen that included mitoxantrone, vorinostat, and bortezomib. 4 patients had a complete response (CR), 1 patient had a partial response (PR) and 1 patient had stable disease for an overall response rate of 5/6 (83%). Clinical trials are in development to assess this combination further for both relapsed MLLr disease as well as newly diagnosed infant ALL. Our data suggests that these three classes of drugs, identified in our laboratory assays, are clinically active thus validating our system. We are now using this platform to proceed with a high throughput drug screen to identify additional compounds for future clinical development. DisclosuresOff Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.

HMGA2 As a Potential Molecular Target in MLL-AF4 Positive Infant Acute Lymphoblastic Leukemia

Leukemic cells of acute lymphoblastic leukemia (ALL) in infants are frequently characterised by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion gene. Among more than 50 genes which have been identified as the fusion partner of the MLL gene, fusion with AF4 is characteristically observed in infant ALL representing a hallmark of poor prognosis. Although recent progress of intensive chemotherapy with or without stem cell transplantation has improved its treatment outcome, the treatment is often accompanied by long-term side effects. Less toxic molecular targeting therapies are therefore necessary for infant ALL.We have previously reported that in infant ALL with MLL fusion gene, microRNA let-7b is significantly downregulated by DNA hypermethylation of its promoter region. The downregulation of let-7b is one of the consequences of oncogenic MLL fusion proteins contributing to leukemogenesis possibly through upregulation of let-7b-regulated target genes with oncogenic potential such as high mobility group AT-hook 2 (HMGA2). HMGA2 is a chromatin-remodelling factor, which alters chromatin architecture by binding to AT-rich regions in the DNA, either promoting or inhibiting the expression of its target genes. One of the targets of HMGA2 is CDKN2A gene which encodes 2 cell cycle regulators p16INK4A and p14ARF. This let-7b-HMGA2-CDKN2A axis regulates cellular growth and senescence of stem cells both in normal and pathological state such as cancer.We initially examined the expression of HMGA2 in leukemic cells obtained from 35 MLL-rearranged infant ALL patients (MLL-AF4, n = 26; MLL-AF9, n = 4; MLL-ENL, n = 5) using quantitative RT-PCR. As results, HMGA2 was highly expressed in most of the patients with MLL fusion gene, especially in MLL-AF4-positive cases, compared to those without the fusion. These results indicate that deregulation of let-7b-HMGA2 axis by MLL fusion may contribute to leukemogenesis and could be a possible target of molecular therapy against MLL-rearranged ALL.As let-7b is downregulated by promoter hypermethylation, demethylating agents such as 5-azacytidine could be applied to recover the expression of the gene in leukemic cells with MLL fusion gene. To test this possibility, leukemic cell lines with MLL-AF4 fusion gene were used. The administration of 5-azacytidine alone was able to restore the expression of suppressed let-7b as well as p16INK4A gene in the leukemic cells, but the effects was incomplete, showing persistent partial promoter methylation. In addition, the recovered expression disappeared when 5-azacytidine was removed. On the other hand, when HMGA2 inhibitor was combined with 5-azacytidine, the expression of let-7b was upregulated and sustained resulting in suppression of HMGA2 protein itself. This upregulation of let-7b and suppression of HMGA2 protein persisted even after the removal of 5-azacytidine, possibly through maintaining of the demethylating status by HMGA2 inhibitor. Inhibition of HMGA2 by either siRNA or HMGA2 inhibitor suppressed the growth of MLL-AF4-positive leukemic cells when analysed by MTT assay. The effects of HMGA2 inhibitor on cell growth inhibition became more prominent in combination with demethylating agent 5-azacytidine.Our results revealed the functional significance of let-7b and HMGA2 in controlling MLL-AF4-positive leukemic cell growth and the therapeutic potential of combining demethylating agent and the HMGA2 inhibitor in the treatment of MLL-AF4-positive ALL. DisclosuresNo relevant conflicts of interest to declare.

Analyses of a Pair of Concordant Twins with Infant ALL and Discordant Clinical Outcome Reveals Immunoescape As a Mechanism of Disease Persistence in MLL-Rearranged Leukemia

MLL-fusion is the most common genetic abnormality in acute lymphoblastic leukemia (ALL) of infancy and occurs in approximately 80% of the cases. Infant ALL represents a biologically distinctive entity with a highly immature pro-B immunophenotype associated with a particularly unfavorable prognosis due to a high proportion of early relapses. This may be due to the survival of dormant residual disease protected by the bone marrow niche.We have characterized a pair of monozygotic twin sisters diagnosed with ALL in early infancy. Tumor cells from both children carried the t(11;19) translocation (MLL-ENL fusion) and both patients were treated according to the ALL-BFM 2000 protocol. Despite good initial treatment responses in both twins, one sister developed very early relapse (twin A) and succumbed to the disease. The other went into continuous complete remission (twin B) and, as of today, is alive after 9 years. The clinical data of the 2 patients are presented in Table 1.Diagnostic bone marrow (BM) aspirates of both sisters were injected into the femoral bones of NOD SCID gamma (NSG) mice. Survival of xenografted mice bearing twin A was significantly longer due to a lower proliferative capacity of twin A as compared to twin B cells. In vivo Bromodeoxyuridine (BrdU) assays revealed that twin B cells were proliferating equally fast in BM and spleen, while in twin A cells, the BM markedly suppressed entry into S-phase. Flow cytometry from xenograft BM identified a large CD34+ population in twin A cells that was almost absent in twin B cells. Furthermore, BM of twin A xenografts showed a CD34+/CD38-/CD19- stem cell like population undetectable in twin B animals. Most interestingly, injection of minimal residual disease (MRD) negative remission bone marrow (PCR for Ig-gene rearrangements and the MLL-fusion gene) of both sisters into NSG mice resulted in a full-blown xenograft leukemia in animals bearing twin A but not twin B. Taken together, these data suggest that fatal relapse in twin A may be due to a quiescent stem cell like population kept in check by unknown mechanisms.Next, we performed gene expression analyses on xenografted leukemias from twin A (initial, leukemia amplified from remission BM, relapse) and from twin B (initial). STRING analysis of gene expression differences revealed that, amongst other findings related to cell cycle regulation and DNA-repair, twin A cells downregulated genes indicating a reduced interferon pathway activity (CXCL10, IFI30, TRAIL, STAT1, OAS1, MX1) and several genes connected to TYRO protein kinase binding protein (TYROBP) shown previously to regulate the activity of natural killer (NK) cells. This suggests that twin A cells may evade immunosurveillance as a mechanism of disease persistence. 51Chromium-release assays with IL-2 stimulated allogeneic NK cells from two healthy donors and the xenografted twin cells showed that twin A cells were significantly less sensitive to NK cell mediated lysis as compared to twin B cells. Whether the tumor cells also display differential susceptibility to antibody-dependent cell-mediated cytotoxicity is currently investigated.While the extensive genomic characterization of the twin leukemias is under way, we show evidence from xenograft experiments, gene expression profiles and functional in vitro experiments that dormant residual cells in MLL-rearranged ALL can evade immunosurveillance. These findings serve as a rationale to employ immunotherapeutic approaches in infant ALL patients in order to improve their dismal prognosis. Finally, we report the first monozygotic twin pair with MLL-rearranged ALL and discordant clinical outcomes. This provides a unique opportunity and a model to gain insight into the clonal composition of infant leukemia and the origins of leukemia relapse.Table 1:Patient characteristics of the MLL-ENL positive twin pair. WBC, white blood cells; BM, bone marrow; CNS, central nervous system; MRD, minimal residual disease.ParameterTwin ATwin BAge at diagnosis (days)98147WBC (initial)/µl334.000103.000Blasts (Peripheral Blood) %9791Blasts (BM) %9897CNS involvementNot determinedNegativeImmunophenotypePro-BPro-BCytogeneticst(11;19)t(11;19)Prednisone-ResponsePoorGoodBlasts (BM), day 15 %321MRD day 33NegativeNegativeMRD day 78Negative<10-4 *Event-free survival227 days>9 yearsTime to relapse227 daysn/aOverall survival390 days>9 years*low positive, not quantifiable DisclosuresNo relevant conflicts of interest to declare.

Favorable Outcome of Hematopoietic Stem Cell Transplantation Using a Targeted Once-Daily Intravenous Busulfan–Fludarabine–Etoposide Regimen in Pediatric and Infant Acute Lymphoblastic Leukemia Patients

Conditioning regimens for pediatric acute lymphoblastic leukemia (ALL) usually include total body irradiation (TBI), but TBI may result in serious sequelae. Busulfan and cyclophosphamide have been used as an alternative to TBI. Etoposide also has been widely used to enhance antileukemic effect. However, toxicities have been reported in some studies using busulfan, cyclophosphamide, and etoposide regimen. A reduced toxicity myeloablative regimen using busulfan and fludarabine showed promising results. Also, therapeutic drug monitoring (TDM) and administration of targeted doses of busulfan have been recommended to improve the outcome of hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the outcome of HSCT using a targeted once-daily i.v. busulfan–fludarabine–etoposide (BuFluVP) regimen in pediatric and infant ALL. Busulfan (age ≥ 1 year, 120 mg/m2; age < 1 year, 80 mg/m2) was administered once daily as the first dose on day −8, and a targeted dose of busulfan was used according to the TDM results on days −7 to −5. Forty-four patients were evaluated. Donor-type neutrophil engraftment was achieved in all patients. Veno-occlusive disease occurred in 7 patients (15.9%), but all patients were successfully treated. Cumulative incidence of treatment-related mortality and relapse were 9.1% and 9.9%, respectively. One-year overall survival and event-free survival rates of all patients were 86.2% and 83.8%, respectively. Twelve patients (27.3%) were infants at diagnosis, and their 1-year overall survival rate was 83.3%. Our study demonstrated that HSCT using a targeted once-daily i.v. BuFluVP regimen showed favorable outcomes and could be an option for HSCT in pediatric and infant ALL.

Favorable Outcome Of Hematopoietic Stem Cell Transplantation Using a Targeted Once-Daily IV Busulfan Fludarabine Etoposide Regimen In Pediatric and Infant ALL Patients

Conditioning regimens for pediatric acute lymphoblastic leukemia (ALL) usually include total body irradiation (TBI), but TBI may result in serious sequelae such as growth impairment, endocrinologic and metabolic problem, and secondary malignancies. Busulfan based conditioning regimen with cyclophosphamide has been used as an alternative to TBI based regimen in many diseases including pediatric ALL. Etoposide has been widely used in hematopoietic stem cell transplantation (HSCT) for lymphoid and myeloid malignancy because of its anti-leukemic effect, and a conditioning regimen containing busulfan, cyclophosphamide and etoposide (BuCyVP) was used in many studies including pediatric patients. But, toxicities have been reported in some studies using BuCyVP regimen. Recently, a reduced toxicity myeloablative regimen using busulfan and fludarabine showed promising results. Also, therapeutic drug monitoring (TDM) and administration of targeted dose of busulfan has been recommended to improve the outcome of HSCT. In this study, we evaluated the outcome of HSCT using a targeted once-daily intravenous (IV) busulfan fludarabine etoposide (BuFluVP) regimen in pediatric and infant ALL. The conditioning regimen was composed of busulfan, fludarabine (40 mg/m2 once daily i.v. on days -8 ∼ -3) and etoposide (20 mg/kg once daily i.v. on days -4 ∼ -2). Busulfan (≥ 1 year-120 mg/m2 and &lt; 1 year-80 mg/m2) was administered once daily as the first dose on day -8, and targeted dose of busulfan was used according to the TDM results on day -7 ∼ -5. A total of 33 patients were evaluated. Donor-type neutrophil engraftment was achieved in all patients. Veno-occlusive disease occurred in 5 (15.2%) patients, but all of them were successfully treated. Grade II-IV acute GVHD developed in 11 patients (grade 2 in 9 patients, grade 3 in 1 and grade 4 in 1 patient) and the cumulative incidence was 33.6%. Chronic GVHD developed in 3 patients (2 extensive and 1 limited). Three patients died of treatment-related mortality (TRM) and the cumulative incidence was 9.1%. The causes of TRM were adenoviral pneumonia in 1, respiratory syncytial viral (RSV) pneumonia in 1 and interstitial lung disease in 1 patient. Relapse occurred in 3 patients and cumulative incidence was 9.1%. Overall survival (OS) and event free survival (EFS) of all patients were 84.9% and 81.8%, respectively. EFS showed no difference according to the type of HSCT (77.8% in related BMT/PBSCT, 82.4% in unrelated BMT/PBSCT, and 85.7% in CBT, P=0.95). Eleven patients (33.3%) were infants at diagnosis, and OS of these patients was 81.8%. Our study demonstrated that HSCT using a targeted once-daily IV BuFluVP regimen showed favorable outcome and could be one option for HSCT in pediatric and infant ALL. Disclosures: No relevant conflicts of interest to declare.

Chemical genomic screening identifies LY294002 as a modulator of glucocorticoid resistance in MLL-rearranged infant ALL

Successful treatment results for MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants remain difficult to achieve. Significantly contributing to therapy failure is poor response to glucocorticoids (GCs), like prednisone. Thus, overcoming resistance to these drugs may be a crucial step towards improving prognosis. We defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples. In the current study, we applied Connectivity Map analysis to perform an in silico screening for agents capable of reversing the prednisolone-resistance profile and induce sensitivity. These analyses revealed that LY294002, a PI3K inhibitor, would potentially fulfill this task. Subsequent validation experiments demonstrated that indeed LY294002, and other known PI3K inhibitors, markedly sensitized otherwise resistant MLL-rearranged ALL cells to prednisolone in vitro. Using quantitative RT-PCR analyses, we validated the modulating effects of the PI3K inhibitors on the expression of the genes present in our prednisolone-resistance profile. Interestingly, prednisolone-sensitizing actions may be mediated by inhibition of FCGR1B. Moreover, only high-level expression of FCGR1B showed to be predictive for a poor prognosis and shRNA-mediated knock-down of FCGR1B led to in vitro prednisolone sensitization. Thus, implementing FDA-approved PI3K inhibitors in current treatments may potentially improve the GC response and prognosis in patients with MLL-rearranged ALL.

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211-8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.

Src kinase-induced phosphorylation of annexin A2 mediates glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia

MLL-rearranged infant acute lymphoblastic leukemia (ALL) (<1 year of age) are frequently resistant to glucocorticoids, like prednisone and dexamethasone. As poor glucocorticoid responses are strongly associated with therapy failure, overcoming glucocorticoid resistance may be a crucial step towards improving prognosis. Unfortunately, the mechanisms underlying glucocorticoid resistance in MLL-rearranged ALL largely remain obscure. We here defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples, demonstrating that, among other genes, high-level ANXA2 is associated with prednisolone resistance in this type of leukemia. Further investigation demonstrated that the underlying factor of this association was the presence of Src kinase-induced phosphorylation (activation) of annexin A2, a process requiring the adapter protein p11 (encoded by human S100A10). shRNA-mediated knockdown of either ANXA2, FYN, LCK or S100A10, all led to inhibition of annexin A2 phosphorylation and resulted in marked sensitization to prednisolone. Likewise, exposure of prednisolone-resistant MLL-rearranged ALL cells to different Src kinase inhibitors exerting high specificity towards FYN and/or LCK had similar effects. In conclusion, we here present a novel mechanism of prednisolone resistance in MLL-rearranged leukemias, and propose that inhibition of annexin A2 phosphorylation embodies a therapeutic strategy for overcoming resistance to glucocorticoids in this highly aggressive type of leukemia.

A Translocation t(5;15)(q15;q11-13) Infant Case with Acute Lymphoblastic Leukemia and Literature Review

Infant acute lymphoblastic leukemia (ALL) represents poor prognosis despite intensive chemotherapy. Rearrangements of chromosome 11q23 are not observed in 34% of the cases. Infant ALL patients with t(5;15)(p15;q11-13) are rare and sporadic. In large series of infant ALL studies, 6 patients have been reported. We present a new case of an infant ALL patient with t(5;15)(p15;q11-13), and a literature review. Considering the data provided by our case and previous reports, we reinforce that this chromosomal abnormality is characteristic of ALL patients under 12 months of age sharing break point in 5p15 and 15q11-13 and strengthen the existence of an infant ALL subgroup characterized by pre-B L1 ALL, CD10-positive, complete remission (100%), and event-free survival (71%), with a relatively good prognosis and clearly less severe than the 11q23 rearrangement cases. This abnormality can be considered a recurrent abnormality on this nosologic group.

Elevated S100A8/S100A9 expression causes glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca(2+) fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca(2+) and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca(2+) levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca(2+) fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.

ANGIOPOIETIN1 - a Novel Factor Implicated In MLL-Rearranged Acute Lymphoblastic Leukemia and Regulated In a Fusion Gene-Dependent Manner

AbstractAbstract 66 Background:MLL-rearranged acute lymphoblastic leukemia (ALL) is prevalent in infants, constituting 70% of the cases. The preferred MLL translocation partner is the gene AF4, resulting in t(4;11)(q21;q23), which arises in 50% of infant ALL patients. This translocation generates the fusion genes MLL/AF4 and AF4/MLL, and is associated with an aggressive clinical presentation and poor outcome. Biologically, cells expressing MLL/AF4 show resistance to stress- and chemotherapy-related apoptosis. Concordantly, we have previously shown that RNAi-mediated depletion of MLL/AF4 in the t(4;11)-positive ALL cell line SEM results in induction of cell death and impaired both clonogenicity and in vivo engraftment. In order to characterize this phenotype on a molecular level, we have performed gene expression profiling of SEM cells depleted of MLL/AF4 and corresponding controls. Expression of >1000 genes was affected, including a subset of angiogenic genes, most prominently ANGIOPOIETIN1 (ANGPT1), a proangiogenic cytokine reported to play a role in acute myeloid leukemia (AML), hematopoietic stem cell (HSC) quiescence and bone marrow (BM) niche maintenance, but to date not implicated in ALL. Here we report a novel link between ANGPT1 expression and MLL-rearranged ALL. Methods:Gene expression profiling was performed using the Illumina HT-12 platform and data processed using BeadStudio and Genespring software suites. ANGPT1 expression was analyzed by real-time RT-PCR (qRT-PCR), and ANGPT1 protein secretion determined using enzyme-linked immunosorbent assay (ELISA). The MLL/AF4 status of cells was modulated with fusion transcript-specific siRNAs and knockdown monitored by qRT-PCR. RNAi-mediated depletion of ANGPT1 was achieved using siRNA or lentiviral shRNA constructs, and validated on transcript and protein level. Effects on cell cycle progression and proliferation in response to ANGPT1 knockdown in t(4;11)-positive cells were assessed by flow cytometry and trypan blue exclusion assay, respectively. For in vivo studies, SEM cells were sequentially transduced to express both luciferase and either non-target control shRNA (shNTC) or shANGPT1. Doubly transduced cells were selected for and FACS-sorted prior to intrafemoral transplantation into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Disease progression was monitored using bioluminescence imaging and engraftment assessed by flow cytometry at the terminal timepoint. Results:ANGPT1 expression was screened in a MLL-rearranged ALL patient cohort (n=35), comprising t(4;11)-positive (n=20), t(11;19)-positive (n=10) and t(9;11)-positive patients (n=5), and in a MLL-wildtype BCP ALL patient cohort (n=8). MLL-rearranged patients showed ANGPT1 upregulation, t(4;11)-positive patients having the strongest overexpression by 232-fold when compared to ANGPT1 levels in CD19+ peripheral blood (PB) cells. A 27-fold and 13-fold upregulation was detected in t(11;19)- and t(9;11)-positive patients, respectively. Conversely, MLL-wildtype BCP ALL patients had similar ANGPT1 levels as CD19+ PB cells, with only a 2-fold increase. In addition to its high expression in t(4;11)-positive ALL, ANGPT1 levels were shown to be dependent on MLL/AF4; a reduction of ANGPT1 mRNA and protein correlated with siRNA-mediated MLL/AF4 depletion in a time-dependent manner in both cell lines and primary patient samples. This was concordant with expression array data, which indicated an up to 4-fold decrease of ANGPT1 in response to MLL/AF4 depletion. The functional role of ANGPT1 in t(4;11)-positive ALL was assessed by RNAi; sustained depletion of ANGPT1 in SEM cells resulted in cell cycle arrest and a marked decrease in proliferation. In vivo, mice transplanted with shANGPT1 expressing SEM cells showed reduced splenic infiltration and development of solid tumours at the injection site, as opposed to a systemic spread of the disease and massive splenomegaly in mice injected with shNTC expressing SEM cells. Conclusions:In this study we have identified ANGPT1 as a novel player in t(4;11)-positive ALL, as defined by overexpression, MLL/AF4-dependent regulation and functional consequences in vivo and in vitro. Currently we are investigating ANGPT1-mediated signalling in t(4;11) ALL cells, as it represents an attractive potential therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Connectivity Mapping Identifies HDAC Inhibitors as Suitable Candidates for the Treatment of t(4;11)-Positive Acute Lymphoblastic Leukemia In Infants

AbstractAbstract 3222To date, MLL-rearranged infant Acute Lymphoblastic Leukemia (ALL) remains the most aggressive type of childhood leukemia characterized by a high rate of early relapses and a grim prognosis. This type of leukemia arises from chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene. The genesis of such translocations seems to be initiated in utero. Hence, MLL-rearranged ALL is typically diagnosed in infants at or shortly after birth. The most common MLL translocation among infant ALL patients, occurring in about 50% of the cases, is t(4;11) generating the oncogenic fusion product MLL-AF4.MLL-rearranged ALL is distinguishable from other ALL subtypes by unique gene-expression profiles. While inappropriate gene activation seems to be largely driven by specific histone modifications, we recently showed that gene inactivation in MLL-rearranged infant ALL can be explained by aberrant promoter hypermethylation. Focusing on genome-wide DNA methylation, we here uncovered that MLL-rearranged infant ALL is characterized by the activation of particular set of (proto-onco)genes as a result of abnormal promoter hypomethylation.In search for therapeutic agents capable of targeting these potential cancer-promoting genes, we applied connectivity mapping on a gene expression signature based on the genes most significantly hypomethylated in t(4;11)-positive infant ALL as compared with healthy bone marrows. Connectivity map analyses revealed that histone deacetylase (HDAC) inhibitors represent the most suitable compounds to reverse this gene signature. We show that six different HDAC inhibitors effectively induce leukemic cell death in primary t(4;11)-positive infant ALL cells in vitro. These cytotoxic responses were accompanied by down-regulation of MYC, SET, RUNX1 and RAN, and were preceded by degradation of the oncogenic MLL-AF4 fusion product.Our data shows that DNA methylation profiles allow the identification hypomethylated proto-oncogenes, and underlines the essential role for epigenetic de-regulation in MLL-rearranged ALL. Furthermore, we show for the first time that connectivity mapping can indirectly be applied on DNA methylation patterns, providing a rationale for HDAC inhibition in t(4;11)-positive leukemias. Given the presented potential of HDAC inhibitors to target important proto-oncogenes including the leukemia-driving MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials. Disclosures:No relevant conflicts of interest to declare.

Specific promoter methylation identifies different subgroups of MLL-rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options

AbstractMLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.

High Expression of the Ca2+-Binding Proteins S100A8 and S100A9 Cause Glucocorticoid Resistance in MLL-Rearranged Infant Acute Lymphoblastic Leukemia.

Abstract 729MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) represents an aggressive and difficult to treat type of leukemia, displaying cellular resistance to several chemotherapeutics, especially to glucocorticoids like prednisolone. As prednisolone response is highly predictive for clinical outcome, it is of utmost importance to unravel the mechanism underlying resistance to this drug.To gain insights in the prednisolone resistance mechanism, we compared gene expression profiles (Affymetrix HU133plus2) from prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patients. This gene signature revealed that multiple genes involved in calcium signaling were up-regulated in prednisolone-resistant samples. Most pronounced up-regulation was observed for the S100 protein family members S100A8 and S100A9, which are calcium-binding proteins, that function in a complex. Quantitative RT-PCR (TaqMan) analyses confirmed that S100A8 and S100A9 mRNA expression was ∼100-fold higher in prednisolone-resistant cells compared to patients sensitive to this drug (p=0.008).Glucocorticoids are known to induce apoptosis by releasing Ca2+ from the endoplasmic reticulum (ER) into the cytosol and towards the mitochondria. Within mitochondria, elevated Ca2+ levels induce cytochrome c release, triggering apoptosis. Since S100A8/A9 are capable of binding free cytosolic Ca2+, we hypothesized that over-representation of these cytosolic proteins may prevent Ca2+ to reach the mitochondria and forestall apoptosis.To test our hypothesis, we first co-incubated prednisolone-sensitive MLL-rearranged ALL cells with prednisolone and EGTA or BAPTA-AM. Both agents represent Ca2+chelators and scavenge free cytosolic Ca2+, thereby mimicking the Ca2+binding by S100A8/A9. Flow cytometry analyses showed that both EGTA and BAPTA-AM inhibited the free cytosolic calcium released by prednisolone. Cytotoxicity tests demonstrated that these prednisolone-sensitive cells became more resistant to prednisolone after co-incubation with either EGTA or BAPTA-AM. Next we asked whether enforced over-expression (using retroviral expression vectors) of S100A8 and/or S100A9 in prednisolone-sensitive MLL-rearranged ALL cells could also inhibit free cytosolic Ca2+and induce prednisolone resistance. Indeed, both S100A8 and S100A9 were capable of binding free cytosolic Ca2+ released by prednisolone, accompanied by a 30-40% increase in cell survival when either S100A8 or S100A9 were over-expressed alone. Simultaneous over-expression of both S100A8 and S100A9 almost completely reversed the prednisolone-sensitive into a prednisolone-resistance phenotype.In conclusion, these findings implicate that high expression of S100A8 and S100A9 contributes to prednisolone resistance in MLL-rearranged infant ALL. Disclosures:No relevant conflicts of interest to declare.

Specific Promoter CpG Island Methylation Patterns Identify Different Subgroups of MLL - Rearranged Infant Acute Lymphoblastic Leukemia, and Define Clinical Outcome

At present, long-term survival rates in childhood acute lymphoblastic leukemia (ALL) easily exceed 80%. However, the prognosis for infants (<1 year) with ALL barely reaches 50%. Infant ALL is characterized by chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene that occur in about 80% of the cases. The most frequent MLL translocations in infant ALL include t(4;11), t(11;19) and t(9;11). In about 20% of the infant ALL cases no MLL rearrangements are observed. Recent gene-expression profiling characterized MLL-rearranged ALL as a unique type of leukemia, that is genetically clearly separable from other ALL subtypes. As epigenetic modifications affect gene-expression, we hypothesized that the specific gene-expression profiles associated with MLL-rearranged ALL may well be driven by epigenetic changes. The best-studied epigenetic event in hematological malignancies constitutes the transcriptional silencing of (tumor suppressor) genes by promoter CpG island hypermethylation. To explore the DNA methylation patterns underlying MLL-rearranged infant ALL, we applied Differential Methylation Hybridization (DMH) using both 9k (Huang, 2002) and 244k CpG island microarrays (Agilent) on primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17), t(9;11) (n=6) or wild-type MLL genes (n=13). The resulting DNA methylation patterns were compared with the patterns found in healthy pediatric bone marrow samples (n=8). In addition, relapse material from three infants with MLL-rearranged ALL was included and compared with the corresponding patient sample obtained at diagnosis. Both CpG island microarray platforms demonstrate that t(4;11) and t(11;19) characterize extensively hypermethylated leukemias, whereas t(9;11)-positive and translocation-negative infant ALL epigenetically resemble normal bone marrow. When the CpG array data (Agilent) were compared with available gene expression profiles (Affymetrix), we found that 95% of the genes from the top 100 of genes most significantly hypermethylated in t(4;11)- or t(11;19)-positive infant ALL were indeed down-regulated. Using the t(4;11)-positive cell line models SEMK2 and RS4;11, we demonstrate that the majority of these hypermethylated genes could be demethylated by the demethylating agent zebularine. Among t(4;11)- and t(11;19)-positive infant ALL samples, two subgroups could be identified displaying either more or less pronounced methylation patterns. Heavy methylation appeared to be associated with a significantly reduced relapse-free survival (p=0.03). Encouraged by these data, we analyzed relapse samples from t(4;11)- and t(11;19)-positive infant ALL patients, and found that these samples were even more extensively hypermethylated than the corresponding initial infant ALL samples. We here present, for the first time to our knowledge, a global view of the methylome in infant patients with MLL-rearranged ALL. We demonstrate that severe promoter CpG hypermethylation is present in t(4;11)- and t(11;19)-positive infant ALL. Of main therapeutic interest is our finding that the degree of DNA methylation among t(4;11)- and t(11;19)-positive infant ALL patients is related to relapse-free survival. Therefore, MLL-rearranged infant ALL patients with heavily methylated leukemias in particular should be considered candidates for therapies including inhibitors of DNA methylation in order to reverse the malignant phenotypes of these leukemias, and improve prognosis. Since MLL-rearranged infant ALL patients are even more hypermethylated at relapse, inhibition of aberrant DNA methylation might also be of vital importance at this stage of disease. Based on these data, we propose to initiate clinical trials using demethylating agents for patients with relapsed MLL- rearranged infant ALL. Meanwhile, we are investigating the in vitro cytotoxicity of various demethylating agents in our laboratory to pave the way for future clinical trials.

Rearranged Infant Acute Lymphoblastic Leukemia.

MLL rearranged Acute Lymphoblastic Leukemia (ALL) represents an unfavorable and difficult to treat type of leukemia that often is highly resistant to glucocorticoids like prednisone and dexamethasone. As the response to prednisone largely determines the clinical outcome of pediatric ALL patients, overcoming resistance to these drugs may be an important step towards improved prognosis. Here we compared gene expression profiles between prednisone-resistant and prednisone-sensitive pediatric ALL patients to obtain gene expression signatures associated with prednisone resistance for both childhood (>1 year of age) and MLL rearranged infant (<1 year of age) ALL. Merging both signatures in search for overlapping genes associated with prednisone resistance in both patient groups we, found that elevated expression of MCL-1 (an anti-apoptotic member of the BCL-2 protein family) appeared to be characteristic for both prednisone-resistant ALL samples. To validate this observation, we determined MCL-1 expression using quantitative RT-PCR in a cohort of MLL rearranged infant ALL samples (n=23), and confirm that high-level MCL-1 expression significantly confers glucocorticoid resistance both in vitro and in vivo. Finally, down-regulation of MCL-1 in prednisone resistant MLL rearranged ALL cells by RNA interference (RNAi) markedly sensitized these cells to prednisone. Therefore we conclude that MCL-1 plays an important role in glucocorticoid resistance and that MCL- 1 suppressing agents co-administered during glucocorticoid treatment may be beneficial especially for MLL rearranged infant ALL patients.

Distinct Methylation Patterns among Infants with MLL Rearranged Acute Lymphoblastic Leukemia.

Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) is characterized by a high incidence of rearrangements of the MLL gene (∼80%) which is associated with a poor prognosis. The most frequent MLL rearrangements in infant ALL are translocations t(4;11), t(11;19) and t(9;11). Recently, gene expression profiling has established MLL rearranged leukemia as a unique type of leukemia (denoted MLL), that is clearly distinguishable from other ALL subtypes. Currently, these gene expression profiles are slowly revealing important genetic properties underlying this aggressive type of leukemia, however, any epigenetic data on MLL are still lacking. Therefore, the present study was designed to unravel the MLL-specific methylation patterns underlying infant MLL by applying differential methylation hybridization (DMH) using CpG island microarrays containing ∼9000 CpG island probes, in duplicate. Primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17) and t(9;11) (n=6) were compared to infant ALL (n=13) and non-infant pediatric ALL (n=15) samples without MLL rearrangements. In addition, healthy pediatric bone marrow samples (n=9) were included as a reference. Compared to healthy controls, 656 CpG island probes were identified as significantly hypermethylated in t(4;11) positive samples, and 131 CpG island probes in t(11;19) positive samples (p<0.01, false discovery rate <5%). Interestingly, t(11;19) positive ALL patients shared 95% of their methylated probes with t(4;11) patients, suggesting a common methylation pattern which is completely absent in both infant and non-infant ALL patients lacking MLL rearrangements. Remarkably, displaying only a single probe significantly methylated as compared to healthy bone marrow, this common methylation pattern is also absent in t(9;11) positive ALL patients, indicating that based on genome-wide methylation, these patients represent a distinct entity clearly distinguishable from other MLL subgroups. Moreover, the fact that t(4;11) patients exhibit 532 methylated CpG island probes that were not found to be methylated in t(11;19) patients, demonstrates that these patients also exhibit a t(4;11) specific set of methylated genes. Identification of the genes represented by these CpG island probes and subsequent validation of the results obtained in this study is currently being performed (using pyrosequencing and methylation specific PCR analyses). In conclusion, these data reveal that different types of MLL rearranged infant ALL show distinct genome-wide methylation patterns. Specifically, infant ALL patients carrying t(4;11) and t(11;19) are characterized by severe CpG island hypermethylation, as compared to both t(9;11) positive infant ALL patients, as well as pediatric ALL patients lacking MLL rearrangements. Therefore, t(4;11) and t(11;19) patients in particular may well be suitable candidates for DNA methylation inhibiting therapeutic intervention. Finally, these promising results for the first time provide epigenetic insights into the complex biology of infant MLL, and clearly warrant further investigation currently being performed at our laboratory.

A Novel Approach for Analyzing Gene Expression Profiles Defines Two Distinct Subgroups of t(4;11) Positive Infant Acute Lymphoblastic Leukemia Patients.

Gene expression profiles (GEPs) are becoming increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome for various types of cancer. However, established gene signatures not always appear helpful in understanding underlying disease mechanisms. Therefore, we explored an alternative approach for analyzing GEPs of a group of t(4;11) positive infant acute lymphoblastic leukemia patients (n=15), a highly aggressive type of MLL rearranged leukemia. We developed a method that uses a relational database program in combination with a normalization approach and specific discriminators. For normalization, every GEP of a given t(4;11) positive ALL sample was compared with 3 GEPs of normal bone marrow aspirates derived from healthy donors. Using the GeneChip Analysis Suite 5.0 program for single comparison analysis, the resulting gene lists were then compared with each other and only consistently identified genes (present on all three gene lists) were used for further analysis. This noise reduction decreased the amount of potentially deregulated target genes to about 30–40%. The remaining gene lists represented highly significant target genes that were then incorporated into a relational database program using specific discriminators. These discriminators were:1.upregulation of HOXA9 and HOXA10,2.presence/absence of the AF4-MLL fusion transcripts in addition to the MLL-AF4 fusion, and3.the localization of the genomic breakpoint within the MLL gene.This pilot study led to promising results, surprisingly classifying individual t(4;11) positive ALL patients into two distinct subgroups. Both subgroups share about 80 target genes, but also display particular sets of subgroup specific target genes. Importantly, these identified target genes can directly be linked to biological properties of t(4;11) positive leukemia cells, and therefore, allow important novel insights into this aggressive type of leukemia in infants.

Silencing of the tumor suppressor gene FHIT is highly characteristic for MLL gene rearranged infant acute lymphoblastic leukemia

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (<1 years of age). Since current chemotherapy fails in >50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (P< or =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL.

MLL gene rearrangements have no direct impact on Ara-C sensitivity in infant acute lymphoblastic leukemia and childhood M4/M5 acute myeloid leukemia

The antimetabolite cytosine arabinoside (Ara-C) is a deoxycytidine analog that is used as a therapeutic agent in many leukemia treatment regimens. In combination with anthracyclines, Ara-C is the most effective agent in the treatment of acute myeloid leukemia (AML). In the treatment of acute lymphoblastic leukemia (ALL), the use of Ara-C is limited. However, leukemic cells from infants (<1 year of age) with ALL, which are resistant to several chemotherapeutic drugs, are in vitro more sensitive to Ara-C as compared to older children with ALL.1, 2 This observation suggested that infant ALL might resemble a subclass of childhood ALL, which may benefit from intensified treatment with Ara-C to improve the dismal prognosis for these patients who to date experience an event-free survival (EFS) of 35%. Therefore, in 1999, a novel collaborative treatment protocol (INTERFANT-99) was designed with intensified use of Ara-C, in order to provide a more specific treatment to infant ALL patients.