Infant Acute Lymphoblastic Leukemia Cells Research Articles

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Preclinical efficacy of azacitidine and venetoclax for infant KMT2A-rearranged acute lymphoblastic leukemia reveals a new therapeutic strategy

Infants with KMT2A-rearranged B-cell acute lymphoblastic leukemia (ALL) have a dismal prognosis. Survival outcomes have remained static in recent decades despite treatment intensification and novel therapies are urgently required. KMT2A-rearranged infant ALL cells are characterized by an abundance of promoter hypermethylation and exhibit high BCL-2 expression, highlighting potential for therapeutic targeting. Here, we show that hypomethylating agents exhibit in vitro additivity when combined with most conventional chemotherapeutic agents. However, in a subset of samples an antagonistic effect was seen between several agents. This was most evident when hypomethylating agents were combined with methotrexate, with upregulation of ATP-binding cassette transporters identified as a potential mechanism. Single agent treatment with azacitidine and decitabine significantly prolonged in vivo survival in KMT2A-rearranged infant ALL xenografts. Treatment of KMT2A-rearranged infant ALL cell lines with azacitidine and decitabine led to differential genome-wide DNA methylation, changes in gene expression and thermal proteome profiling revealed the target protein-binding landscape of these agents. The selective BCL-2 inhibitor, venetoclax, exhibited in vitro additivity in combination with hypomethylating or conventional chemotherapeutic agents. The addition of venetoclax to azacitidine resulted in a significant in vivo survival advantage indicating the therapeutic potential of this combination to improve outcome for infants with KMT2A-rearranged ALL.

Single-Cell Transcriptomics Reveals Similarity of Aggressive Infant Acute Lymphoblastic Leukemia Cells to Early Hematopoietic Progenitors

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While the prognosis for children diagnosed with ALL has significantly improved in recent decades, the outlook for infants (under 12 months of age) with ALL remains bleak. In particular, patients with a rearrangement in the KMT2A gene (KMT2A-r) frequently relapse and have low event-free survival. Recent work shows that cancer cells from infant ALL patients with KMT2A-r show a high degree of transcriptional similarity to early lymphocyte precursors (ELPs), suggesting a molecular plasticity that may drive the aggressiveness of this cancer. In this study, we extend these findings to patients with germline KMT2A (KMT2A-g) and assess the predictive value of the ELP signature. Methods: 10x Genomics single-cell Multiome (v1 chemistry) sequencing was performed to generate RNA sequencing (scRNAseq) data for samples collected at diagnosis and remission from 34 patients presenting with ALL at < 12 months of age - 25 KMT2A-r patients and 9 KMT2A-g patients. KMT2A-r patients had either KMT2A-AFF1 (n = 12) or KMT2A-MLLT1 (n = 13) fusions. 19/25 KMT2A-r patients and 0/9 KMT2A-g patients subsequently relapsed. Sequencing data were aligned to GRCh38 and counted using cellranger-6.0.1, with downstream analysis performed using Seurat 4.0.2 in R 4.0.3. A logistic regression model from Khabirova et al., 2022, fit on published scRNAseq data from fetal bone marrow cells (Jardine et al., 2021), was subsequently tested on sequencing data to produce similarity scores for each infant ALL cell to cell types along the hematopoietic developmental trajectory. Results: Our findings confirm that cells from infant ALL patients with KMT2A-r show a high degree of transcriptional similarity to ELPs (Figure A). We find that this holds true at diagnosis (DX) regardless of whether the patients later relapsed or not. Moreover, we observe that in KMT2A-g infant ALL patients, cells show a lower degree of similarity to ELPs than their KMT2A-r counterparts, but nonetheless show a greater similarity to ELPs than other hematopoietic cells. We do not observe a high degree of similarity to ELPs at remission (MD) in any patient group, supporting the idea that it is leukemic blasts that are similar to ELPs. Strikingly, we also observe that among KMT2A-r patients, a higher proportion of cells highly similar to ELPs is not predictive of subsequent relapse (Figure B). Conclusions: Here we confirm that KMT2A-r infant ALL cancer cells are transcriptionally similar to ELPs, highly plastic hematopoietic progenitors. This plasticity may play a significant role in the aggressiveness of this cancer. We show that this similarity does not exist to the same extent in KMT2A-g patients, consistent with their better prognosis. Additionally, we demonstrate that proportion of ELP-like cells is not predictive of relapse among KMT2A-r patients, suggesting that more work is needed to better understand the significance of similarity to ELPs in this disease. Deeper investigation of gene expression in ELP-like leukemic blasts will shed further light on the molecular mechanisms of this cancer and facilitate improved treatment selection and development of novel therapies for infants with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Throughput Drug Library Screening in Primary KMT2A-Rearranged Infant ALL Cells Favors the Identification of Drug Candidates That Activate P53 Signaling.

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.

KMT2A-Rearranged Infant Acute Lymphoblastic Leukemia Cells Undergo ER-Stress-Induced Apoptosis Following Exposure to Proteasome Inhibitors

Infants diagnosed with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event free survival of 23-44%. To identify new treatment approaches we previously performed in vitro and in vivo assays to evaluate the activity of FDA approved compounds in 15 primary KMT2Ar infant leukemia samples. Three classes of agents were found to be active in these assays: proteasome inhibitors, anthracyclines, and histone deacetylase inhibitors (HDACi). KMT2Ar infant leukemia samples were exquisitely sensitive to the proteasome inhibitor bortezomib, requiring 10-100 fold less drug to achieve 50% toxicity when compared to non-KMT2Ar childhood ALL. Bortezomib is FDA approved for multiple myeloma and laboratory studies using this model system have previously demonstrated responses to be mediated through several mechanisms including NFKB inhibition, stabilization of cell cycle regulatory proteins, and perhaps most importantly the induction of an unfolded protein response (UPR) and endoplasmic reticulum (ER)-stress-induced apoptosis. To evaluate global protein dynamics in KMT2Ar ALL cells treated with bortezomib, we performed tandem mass tag (TMT) quantitative mass spectrometry on synchronized SEM cells exposed to either 50nM of bortezomib or DMSO at 0 hours (hr), 6hr, 12hr, 16hr, and 20hr. Applying pairwise comparison for 9232 unique proteins measured over the time course compared to untreated controls, we identified 1593 proteins with a log2 fold change &gt;1.5 in bortezomib treated cells compared to 101 proteins in the DMSO control (FDR&lt;0.01). Several proteins associated with ER-stress-induced apoptosis including ATF4, DDIT4, ATF3, TSC22D3 (GILZ), and PMAIP1 (NOXA) were upregulated more than 3-fold between 6 and 20hr, suggesting this pathway may play a role in bortezomib induced apoptosis of KMT2Ar cells (p&lt;0.05 and log2 fold change of +/- 0.58). To validate this finding and further understand the role of the UPR and ER-stress-induced apoptosis, we evaluated seven key mediators of this pathway by western blot following bortezomib exposure on synchronized SEM cells over a 12 hour time course including ATF4, ATF6, CHOP, PERK, GADD34, CReP, and eIF2α as well as phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). This demonstrated a critical time point at 6hr where an increase in ATF4 (3.5 fold), CHOP (1.6 fold), and CReP (2.9 fold) protein levels was accompanied by a decrease in p-PERK (0.7 fold), and p-eIF2α (0.8 fold) whereas GADD34 levels remained constant. Although full-sized ATF6 (ATF6a) protein showed a considerable increase (1.9 fold), the levels of cleaved ATF6 (ATF6f) were only slightly increased (1.2 fold) consistent with ATF4-mediated upregulation of CHOP leading to increased protein synthesis along with ATP depletion, oxidative stress, and cell death. While GADD34 has been shown to be the main phosphatase that functions in a negative feedback loop to resolve cell stress, recent data suggests that stabilization of CReP mRNA by ER stress is able to reverse eIF2α phosphorylation at later stages of UPR leading to re-expression of key UPR proteins. Further, p-eIF2α-attenuated protein synthesis, and not ATF4 mRNA translation has been shown to promote cell survival. Our data support a model whereby the UPR and ER-stress in KMT2Ar ALL cells is induced upon exposure to bortezomib leading to increased levels of ATF4 and CHOP. Attenuation of p-eIF2α by CReP further contributes to cell death through the recovery of protein synthesis in a setting of limited protein folding capacity. These results support the use of proteasome inhibitors in KMT2Ar leukemia which is currently being formally evaluated in a Phase II clinical trial for newly diagnosed patients with infant ALL (NCT02553460). Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target.CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens.Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level.Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot.Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. DisclosuresNo relevant conflicts of interest to declare.

Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia.

The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

MEK inhibition is a promising therapeutic strategy for MLL-rearranged infant acute lymphoblastic leukemia patients carrying RAS mutations.

Acute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence of a RAS mutation decreased the survival chances even further. We hypothesized that targeting the RAS signaling pathway could be a therapeutic strategy for RAS-mutant infant ALL patients. Here we show that the MEK inhibitors Trametinib, Selumetinib and MEK162 severely impair primary RAS-mutant MLL-rearranged infant ALL cells in vitro. While all RAS-mutant samples were sensitive to MEK inhibitors, we found both sensitive and resistant samples among RAS-wildtype cases. We confirmed enhanced RAS pathway signaling in RAS-mutant samples, but found no apparent downstream over-activation in the wildtype samples. However, we did confirm that MEK inhibitors reduced p-ERK levels, and induced apoptosis in the RAS-mutant MLL-rearranged ALL cells. Finally, we show that MEK inhibition synergistically enhances prednisolone sensitivity, both in RAS-mutant and RAS-wildtype cells. In conclusion, MEK inhibition represents a promising therapeutic strategy for MLL-rearranged ALL patients harboring RAS mutations, while patients without RAS mutations may benefit through prednisolone sensitization.

MEK Inhibition Is a Promising Therapeutic Strategy for MLL-Rearranged Infant ALL Patients Carrying RAS Mutations

Background: Acute Lymphoblastic Leukemia (ALL) in infants is characterized by a high incidence (~80%) of chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene, fusing the N-terminal portion of MLL to the C-terminal region of one of its translocation partner genes. MLL-rearranged infant ALL patients are challenged by a very poor prognosis (i.e. 30-40% 5-year EFS), hence the need for better risk stratification and improved therapeutic solutions is evident. We recently screened a relatively large cohort (n=109) infant ALL patients (all enrolled in INTERFANT treatment protocols) for the presence of KRAS and NRAS mutations and found that the incidence of such mutations ranges between 14-24%, depending on the type of MLL translocation. Moreover, these mutations were found to represent independent predictors of exceedingly poor prognosis; patients carrying RAS mutations essentially stand no chance in surviving their malignancy, as all RAS mutation-positive MLL-rearranged infant ALL patients deceased within 3 years from diagnosis.Aims: Here we aimed to identify a therapeutic strategy to improve the prognosis of MLL-rearranged infant ALL patients carrying RAS mutations.Methods: For this, 8 small molecule inhibitors against different RAS-pathway components were selected and initially tested for anti-leukemic activity against the MLL-rearranged ALL cell lines SEM and RS4;11 (RASwt) and KOPN8 (RASmut) using MTS cell viability assays. Next, primary MLL-rearranged infant ALL samples (n=20) all carrying MLL translocation t(4;11) (giving rise to the MLL-AF4 fusion protein) either with (n=6) or without (n=14) RAS mutations were exposed to these inhibitors in MTT cytotoxicity assays. In addition, we assessed the RAS activity in RAS mutated and wild-type MLL-rearranged infant ALL cells, and performed immunoblotting analysis of downstream MEK and ERK both in the absence and presence of the MEK inhibitors.Results: We found that the MEK inhibitors MEK162, Selumetinib and Trametinib effectively reduced the viability of KOPN8 cells (RASmut), whereas SEM and RS4;11 cells (RASwt) largely remained unaffected. In line with this, MLL-AF4+ infant ALL patient samples carrying RAS mutations were significantly more sensitive to these MEK inhibitors when compared with patients carrying wild-type RAS genes: LC50 values for MEK162 were 0.04 vs. 26.9 µM (p<0.01), for Selumetinib 0.04 and 23.7 µM (p<0.01), and for Trametinib 0.01 vs. 26.5 µM (p<0.01), respectively. Furthermore, the presence of RAS mutations in primary MLL-rearranged infant ALL samples was associated with significantly increased RAS activity, as determined by immunoprecipitation of GTP-bound RAS. Remarkably, however, enhanced RAS activation did not manifest itself in the form of increased phosphorylation of the downstream ERK protein, while a slight increase of MEK phosphorylation was observed. Yet, MEK inhibitor exposure in both KOPN8 and SEM cells resulted in nearly complete abrogation of ERK phosphorylation, without affecting total ERK protein levels, suggesting that the loss of ERK activation plays an important role in the observed anti-leukemic effects. Furthermore, MEK162 and Selumetinib seemed to induce accumulation of phosphorylated MEK, while MEK phosphorylation was hardly affected by Trametinib. Interestingly, a subgroup of patient samples (n=5) with wild-type RAS also showed sensitivity towards MEK inhibition, similar to the primary cells with RAS mutations. However, this observation could not be explained by increased RAS activation, nor by the phosphorylation levels of either MEK or ERK.Conclusions: Our data show that MEK inhibition represents a promising therapeutic approach for MLL-rearranged infant ALL patients carrying additional RAS mutations. Furthermore, the mechanism of action provoked by these MEK inhibitors seems to involve abrogation of ERK phosphorylation, but the initial level of ERK phosphorylation did not correlate with MEK inhibitor sensitivity, and has no predictive value. Currently we are in the process of testing the efficacy of the above mentioned MEK inhibitors in vivo using a xenograft mouse model, while further elucidation of the molecular mechanisms underlying the anti-leukemic effects of these inhibitors in MLL-rearranged ALL cells is in progress. DisclosuresNo relevant conflicts of interest to declare.

PI3K/AKT/mTOR Signaling Is a Significant Druggable Pathway In Infant Acute Lymphoblastic Leukemia (ALL)

IntroductionInfant acute lymphoblastic leukemia (ALL) is an orphan disease with unmet need for safe effective therapies. This is an urgent problem because conventional chemotherapies are ineffective and have life-threatening toxicities in infants. Although the MLL rearrangements occurring in 75% of cases are associated with poor outcome, survival is inferior whether MLL is rearranged or not. We recently reported that infant ALL proved sensitive to obatoclax mesylate (GeminX Pharmaceuticals; now an indirect, wholly owned subsidiary of Teva Pharmaceutical Industries Ltd.) in vitro regardless of poor prognostic features including MLL gene rearrangement. Moreover, we showed that the leukemia cell killing by obatoclax involved apoptosis, necroptosis and autophagy (Urtishak et al., Blood 2013). Therefore, the recent pharmaceutical abandonment of obatoclax led us to search for similarly acting drugs, the Results of which identified the well-known antipsychotic thioridazine as a candidate for potential repurposing. MethodsCorrelative analyses were performed between basal gene expression profiles at leukemia diagnosis and single agent obatoclax EC50 values from MTT assays in 47 cases of infant ALL from the Children's Oncology Group P9407 trial (25 MLL-AF4; 8 MLL-ENL; 7 other MLL-rearranged; 7 MLL-germline) in order to find a priori determinants of obatoclax sensitivity; significant genes were further studied by Ingenuity Pathway Analysis (IPA). A search for similarly acting compounds was conducted by Connectivity Map analysis of gene expression profiles of MLL-AF4 ALL cell lines after obatoclax treatment. MTT assays without and with cell death pathway inhibition, Western blot and flow cytometric cell death assays, and phosphoflow cytometric signaling analyses were utilized to investigate activity and target modulation by potential candidates. ResultsIPA identified significant correlations between basal gene expression of the mTOR and downstream intersecting eIF4/p70S6K signaling programs and obatoclax EC50 in all 47 primary cases of infant ALL, as well as in the subset of the 25 cases with MLL-AF4 rearrangements. Consistent with the relevance of this pathway in leukemia cell killing that was suggested by the basal gene expression profiles in the primary cases, the Connectivity Map analysis of obatoclax-treated cell lines for compound matching returned a number of highly ranked PI3K/AKT/mTOR signal transduction inhibitors as potential obatoclax substitutes. Three of the compounds (LY294002, wortmannin, thioridazine) were not only cytotoxic in MLL-AF4 ALL cell lines, but also they abrogated PI3K/AKT/mTOR signaling as indicated by robust inhibition of phosphorylated S6. Of these compounds, the phenothiazine derivative thioridazine, which has been used clinically for decades as a neuroleptic, was of high interest because of potential advantages of drug repurposing for more rapid drug advancement. Moreover, detailed flow cytometric and Western blot analyses, and MTT assays of thioridazine in the presence of cell death pathway inhibitors validated activation of all three cell death mechanisms in the MLL-AF4 ALL cell lines similarly to obatoclax. ConclusionsThioridazine is a well-known antipsychotic drug that also has recently recognized properties as a PI3K/AKT/mTOR signaling inhibitor and as an inhibitor of other pathways relevant to cancer. In MLL-AF4 ALL cell lines characterized by the most common chromosomal translocation in infant ALL, single-agent thioridazine is highly cytotoxic, robustly inhibits PI3K/AKT/mTOR signaling and, moreover, like obatoclax, demonstrates activity as a multi-cell-death pathway agonist. Further preclinical studies now are warranted to determine the extent to which thioridazine inhibits PI3K/AKT/mTOR signaling and causes leukemia cell killing in primary infant ALL cells in vitro and in vivo. The repurposing strategy that this drug may allow could have promise to streamline drug development in infant ALL where the need for new therapies is so urgent. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Potent obatoclax cytotoxicity and activation of triple death mode killing across infant acute lymphoblastic leukemia

AbstractSurvival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Connectivity mapping identifies HDAC inhibitors for the treatment of t(4;11)-positive infant acute lymphoblastic leukemia

MLL-rearranged infant acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia characterized by a unique gene-expression profile. We uncovered that the activation of particular (proto-onco)genes is mediated by 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. This analysis revealed histone deacetylase (HDAC) inhibitors as suitable candidates to reverse the unfavorable gene signature. We show that HDAC inhibitors effectively induce leukemic cell death in t(4;11)-positive primary infant ALL cells, accompanied by downregulation of MYC, SET, RUNX1, RAN as well as the MLL-AF4 fusion product. Furthermore, DNA methylation was restored after HDAC inhibitor exposure. Our data 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-specific MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials.

Mixed Lineage Leukemia Gene Rearrangements - Epigenetic Diseases Require Epigenetic Treatments

AbstractAbstract SCI-8Reaching optimal treatment results in MLL-rearranged Acute Lymphoblastic Leukemia (ALL) remains one of the major challenges in pediatric oncology. This aggressive type of ALL is initiated by chromosomal translocations involving the MLL gene, fusing the N-terminal portion of the MLL gene to the C-terminal region of one of its many translocation partner genes. Such MLL rearrangements generate fusion proteins that possess pronounced transforming capacity and typically arise in utero. Hence, MLL-rearranged ALL is usually diagnosed in infants at or shortly after birth. The survival chances for these young patients still ranges from 20–40%, while the long-term survival rates for childhood ALL in general easily exceeds 80%.MLL-rearranged ALL samples display unique and ample deregulated gene expression profiles that are clearly distinguishable from profiles found in other ALL subtypes. Inappropriate gene activation seems to be largely driven by abnormal histone modifications established by the MLL fusion protein itself. In contrast, we recently showed that substantial amounts of gene inactivation in MLL-rearranged infant ALL can be explained by aberrant promoter hypermethylation. The expression of numerous of these epigenetically silenced genes can be restored by demethylating agents and results in severe induction of leukemic cell death. Moreover, the degree of genome-wide promoter methylation appeared to be of prognostic significance with patients displaying more heavily methylated genomes being at extreme high-risk of disease relapse.In additional studies focusing on genome-wide promoter methylation we found that MLL-rearranged infant ALL is characterized by the activation of a particular set of proto-oncogenes as a result of abnormal promoter hypomethylation. Strikingly, the activation of several of these genes had already been associated with activating histone marks induced by the MLL fusion protein. Together these findings demonstrate the sound interplay between histone modifications and downstream DNA methylation. In search of therapeutic strategies to target these epigenetically activated proto-oncogenes, we found that HDAC inhibitors represent particularly suitable compounds. Exposing MLL-rearranged infant ALL cells to HDAC inhibitors effectively induced leukemic cell death, while at similar concentrations normal bone marrow samples were practically unharmed. Interestingly, these cytotoxic responses were accompanied by the rapid down-regulation of multiple of the proto-oncogenes, but more importantly, was preceded by the degradation of the oncogenic MLL fusion product.Collectively, these observations clearly demonstrate that MLL-rearranged ALL represents an epigenetic malignancy that should be treated accordingly using epigenetic drugs like demethylating agents and HDAC inhibitors. With these pre-clinical studies showing great potential of these drugs in vitro, these compounds should now be tested in vivo in mouse models to pave the way for future clinical trials. 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.

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of <or=0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of £0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL-rearranged ALL cells in a sequence-dependent manner

Mixed lineage leukemia (MLL) rearrangements occur in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL). These cases have a poor prognosis with current therapy. The FLT3 kinase is overexpressed and constitutively activated in MLL-rearranged ALL cells. The FLT3 inhibitor CEP-701 selectively kills these cells, but is unlikely to be curative if used as monotherapy. To identify potentially synergistic combination strategies, we studied CEP-701 and six standard chemotherapeutic agents in three sequences of exposure (S1: chemotherapy followed by CEP-701, S2: simultaneous exposure to both; and S3: CEP-701 followed by chemotherapy) using MLL-rearranged ALL cell lines and patient bone marrow samples. MTT cytotoxicity and annexin V binding apoptosis assays were used to assess antileukemic effects. Combination indices (CI) were calculated for each combination (CI<0.9 - synergistic; CI 0.9-1.1 - additive; CI>1.1 - antagonistic). A striking pattern of sequence-dependent synergy was observed: S1 was markedly synergistic (mean CI=0.59+/-0.10), S2 was additive (mean CI=0.99+/-0.09) and S3 was antagonistic (mean CI=1.23+/-0.10). The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, as these effects are not seen in control cells without activated FLT3.

Combinations of the FLT3 Inhibitor CEP-701 and Chemotherapy Synergistically Kill Infant and Childhood MLL-Rearranged ALL Cells in a Sequence-Dependent Manner.

Rearrangement of the MLL gene at chromosome 11q23, which occurs in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL), confers a dismal prognosis despite maximally intensified chemotherapy and allogeneic transplantation. Novel therapies are clearly needed for these patients. The FLT3 tyrosine kinase represents an attractive potential target in these leukemias, due to the marked overexpression of FLT3 in cases of MLL-rearranged ALL. We have previously shown that MLL-rearranged infant and childhood ALL leukemic cells express high levels of constitutively activated FLT3 receptor protein, and that these cells are selectively killed by exposure to the FLT3 inhibitor CEP-701. However, FLT3 inhibition is unlikely to represent curative therapy for these patients if used as monotherapy. Thus, we examined various combinations of CEP-701 with chemotherapy to look for potentially synergistic therapeutic strategies. We performed MTT cytotoxicity and annexin V binding (AVB) apoptosis assays on MLL-rearranged ALL cell lines (SEM-K2 and HB-1119) and three primary patient samples (all of which we show to express high levels of constitutively activated FLT3) after exposure to multiple dose combinations of CEP-701 and six chemotherapy agents (vincristine, l-asparaginase, dexamethasone, daunorubicin, etoposide and cytarabine). Since we have previously demonstrated that the nature of the interactions between CEP-701 and chemotherapy agents (i.e., synergistic, additive or antagonistic) are sequence-dependent in AML cells, we performed the MTT and AVB assays in three sequences designed to mimic the potential clinical uses of the combinations (Seq1: chemotherapy followed by CEP-701; Seq2: simultaneous exposure to both; and Seq3: CEP-701 followed by chemotherapy). The nature of the interaction between CEP-701 and each chemotherapy agent was determined using the median effect method of Chou and Talalay, which calculates a combination index (CI) for each combination (CI < 0.9 - synergistic; CI 0.9-1.1 - additive; CI > 1.1 antagonistic). A striking pattern of sequence-dependent synergy was observed: Seq1 was markedly synergistic (mean CI +/− SD = 0.59 +/− 0.18), Seq2 was additive (CI = 0.99 +/− 0.29) and Seq3 was antagonistic (CI = 1.23 +/− 0.40). The sequence-dependence is attributable to the G1 cell cycle arresting effects of CEP-701 in these cells. An MLL-rearranged cell line that expresses low levels of minimally activated FLT3 (RS4-11) demonstrates minimal cell cycle arrest when treated with CEP-701, and mere additivity in all 3 CEP-701/chemotherapy combination sequences (Seq1: CI = 0.95 +/− 0.10; Seq2: CI = 1.01 +/− 0.09; Seq3: CI = 1.06 +/− 0.10), confirming that the observed pattern of synergy is mediated through the effects of CEP-701 on FLT3 signaling. These results can help guide the combination of CEP-701 with existing chemotherapy regimens for infants and children with MLL-rearranged ALL, with the goal of improving the dismal outcome for these patients.

Differential mRNA expression of Ara-C–metabolizing enzymes explains Ara-C sensitivity in MLL gene–rearranged infant acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P =.007) and accumulated 2.3-fold more Ara-CTP (P =.011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P =.026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P =.001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r(s) = -0.58, P =.006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n = 14) and germ line MLL cases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P =.001) and a 2.7-fold higher hENT1 (P =.046) mRNA expression in patients with MLL gene-rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL gene-rearranged infant ALL.

Defective Expression of Bruton's Tyrosine Kinase in Acute Lymphoblastic Leukemia

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that serves an essential role in B cell signaling and development. We examined the BTK expression profile of primary leukemic cells from infants with newly diagnosed acute lymphoblastic leukemia (ALL) (N =14) and from pediatric patients with newly diagnosed (N =10) or relapsed (N =5) B-lineage ALL. Analysis of BTK protein and mRNA expression in the infant patient cells (N =14) showed variable levels of BTK expression with the majority of samples having reduced to absent BTK expression. Sequence analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products of Btk mRNA from infant leukemia cells revealed the presence of aberrant transcripts. These Btk transcripts were characterized by either deletion of exon 16 (Δ 16) alone or deletion of both exons 15 and 16 (Δ 15 and 16). These deletions involve exact exon skipping and encode BTK proteins with either a deleted (Δ 16), or truncated (Δ 15 and 16) kinase domain. Extension of these Btk transcript sequencing studies to 15 pediatric B-lineage ALL patients revealed expression of exon 16 deleted Btk transcripts in several pediatric patients, however, none of these pediatric patients expressed transcripts with the exon 15 and 16 deletion. Both reduced expression of Btk message and expression of aberrant deleted Btk transcripts would contribute to reduced BTK protein expression and function in B-lineage leukemia cells. Since BTK is required for radiation induced apoptosis, reduced to absent expression of functional BTK in infant ALL cells could contribute to their radiation resistance.