Target In Acute Lymphoblastic Leukemia Research Articles

Disruption of cotranscriptional splicing suggests RBM39 is a therapeutic target in acute lymphoblastic leukemia

AbstractThere are only a few options for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus, this is a major area of unmet medical need. In this study, we reveal that the inclusion of a poison exon in RBM39, which could be induced by both CDK9 or CDK9 independent cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases (CMGC) kinase inhibition, is recognized by the nonsense-mediated messenger RNA decay pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors led to protein downregulation and the inhibition of ALL growth, particularly in relapsed/refractory B-ALL. Mechanistically, disruption of cotranscriptional splicing by the inhibition of CMGC kinases, including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), led to alteration in the SF3B1 and Pol II association. Disruption of SF3B1 and the transcriptional elongation complex altered Pol II pausing, which promoted the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppressed the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong antitumor activity in preclinical models. Our data reveal that relapsed/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide 2 potential strategies to target this axis.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones >5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Epigenetic activation of the FLT3 gene by ZNF384 fusion confers a therapeutic susceptibility in acute lymphoblastic leukemia

FLT3 is an attractive therapeutic target in acute lymphoblastic leukemia (ALL) but the mechanism for its activation in this cancer is incompletely understood. Profiling global gene expression in large ALL cohorts, we identify over-expression of FLT3 in ZNF384-rearranged ALL, consistently across cases harboring different fusion partners with ZNF384. Mechanistically, we discover an intergenic enhancer element at the FLT3 locus that is exclusively activated in ZNF384-rearranged ALL, with the enhancer-promoter looping directly mediated by the fusion protein. There is also a global enrichment of active enhancers within ZNF384 binding sites across the genome in ZNF384-rearranged ALL cells. Downregulation of ZNF384 blunts FLT3 activation and decreases ALL cell sensitivity to FLT3 inhibitor gilteritinib in vitro. In patient-derived xenograft models of ZNF384-rearranged ALL, gilteritinib exhibits significant anti-leukemia efficacy as a monotherapy in vivo. Collectively, our results provide insights into FLT3 regulation in ALL and point to potential genomics-guided targeted therapy for this patient population.

Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Acute Lymphoblastic Leukemia

Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes.Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro.Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies.We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels.Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. [Display omitted] DisclosuresAbukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

Clinical Significance of Phd Finger Protein 2 As a Novel Ikaros Target in Acute Lymphoblastic Leukemia

Background: PHD finger protein 2 (PHF2) mediates demethylation of dimethylated ‘Lys-9‘ of histone H3 (H3K9me2), followed by transcription activation of target genes. PHF2 is a positive epigenetic regulator and is associated with tumor suppression in several types of cancer. Until now, there have been no reports about the PHF2 expression in leukemia patients and its roles in acute lymphoblastic leukemia (ALL).Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high risk B-progenitor ALL and are associated with poor outcome. However, it is still undetermined whether and how IKAROS regulates PHF2 expression. Methods: The 164 patients with newly-diagnosed ALL (107 B-ALL and 57 T-ALL; 12-77 years old) were recruited between June 2008 and June 2016 at the First Affiliated Hospital of Nanjing Medical University and Zhongda Hospital Southeast University. The study was approved by the Ethics Committee of the two hospitals. Patients were allocated into high or low PHF2 expression cohorts (4th quartile vs . 1st-3rd quartiles) with a cut-off value determined by SPSS 20.0. The genomic amplification combined direct sequencing was used for detection of IKZF1 deletion. PHF2 expression was determined by qPCR in cells . The enrichment of IKAROS binding and H3K4me3 in the promoter of PHF2 was determined by the chromatin-immunoprecipitation assay combined qPCR (qChIP). IKZF1 knockdown was achieved in cells by transfection of human IKZF1 shRNA constructs in the GFP vector using the Neon Transfection System and with scrambled shRNA as a control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using univariate and multivariate Cox models. Relapse-free survival and overall survival were estimated by the Kaplan-Meier method and compared by log-rank test. Statistical analyses used SPSS version 20.0. Results: PHF2 mRNA levels in bone marrow samples from adults with ALL, especially those with B-cell ALL (B-ALL), were significantly lower than those in normals. The B-ALL cohort with low PHF2 mRNA levels was associated with a higher median bone marrow blast percentage (90.4% vs. 82.4%; P=0.001), a higher percentage of stem cell marker CD34+ (82.3% vs. 60.0%; P=0.021), myeloid marker CD33+ (48.5% vs. 25.0%; P=0.046), splenomegaly (46.8% vs. 23.1%, P=0.034) and delayed remission more than 4 weeks (48.7% vs. 19.0%; P=0.015), also a higher frequency of Ik6 (+) (46.3% vs. 8.0%, P=0.001) and a lower median PLT count in comparison to the high PHF2 expression cohort. Moreover, ChIP-seq data identified IKAROS binding peaks in the PHF2 promoter region in Nalm6 B-ALL and primary B-ALL cellsand the bindings were confirmed by the qChIP assay . IKAROS overexpression promotes PHF2 mRNA levels, conversely, IKZF1 knockdown decreased PHF2 expression in Nalm6 and CEM cells. These data indicate a direct effect of IKAROS on PHF2 transcription. The patients with IKZF1 deletion (Ik 6) have significantly lower PHF2 expression (0.03201 ± 0.000527 vs. 0.11331± 0.05773; P=0.033), which suggests that the IKZF1 deletion may contribute to low PHF2 expression in ALL patients. Treatment of Nalm6 and CEM cells with CK2 inhibitor CX-4945, which functions as IKAROS activator, promoted expression of PHF2 mRNA in ALL cell lines and primary cells and IKZF1 knockdown blocks this effect. CX-4945 treatment also increases the binding of IKAROS and H3K4me3 enrichment to the PHF2 promoter region in Nalm6 and CEM ALL cells as well as in primary ALL cells. Conclusion: PHF2 low expression was significantly associated with leukemia proliferation and several poor prognostic indicators in adult ALL patients. IKAROS directly regulates PHF2 transcription, and that CK2 inhibitor promotes PHF2 transcription through histone modification in an IKAROS dependent manner. Our data demonstrates the oncogenic effects of IKAROS dysfunction induced decreases in PHF2 expression. This is the first report to demonstrate that low PHF2 expression is significantly associated with Acute Lymphoblastic Leukemia. We suggest that PHF2 expression levelsmay be integrated into future adult ALL risk-stratification models. DisclosuresDovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees.

Interleukin-2 Receptor Alpha Chain, Also Called CD25, Is a Potential Target in Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is a molecularly heterogeneous disease originating from clonal proliferation of precursor B-lineage cells. In adults, ALL diagnosis is still associated with a dismal prognosis due to the lack of specific targeted therapies. This study was designed to investigate the expression of interleukin-2 receptor alpha chain CD25 in B-ALL and its biological significance, especially following the availability of specific CD25 targeting compounds. Methods:The expression of IL2RA (CD25 gene) was detected by flow cytometry (FC), immunohistochemistry and Western blot analysis, in 25 newly diagnosed ALL patients, both Philadelphia positive (12 patients) and Philadelphia negative (13 patients). Similarly, CD25 expression was assessed in four B-ALL commercially available cell lines. Infection with shRNA specifically directed against CD25 was used to evaluate apoptosis induction and cell cycle arrest in primary B-ALL cells established from two patients. Results:Our data suggest that ALL, and in particular Ph-positive ALL, aberrantly expresses the interleukin-2 receptor alpha chain, CD25. Whereas normal B cells display low amounts of CD25, primary ALL cells and ALL cell lines (over)-express CD25. While the high frequency of CD25 on the surface of many different hematological tumor cells has been established and confirmed in our study, there is little investigation focusing on the significance of CD25 expression. Indeed, CD25 may be present on ALL cells and enable oncogenic signaling pathways. In such respect, we observed that CD25 silencing in primary cells promotes cell cycle arrest and apoptosis induction. While these data support the rational to target CD25, ALL cells did not appear to be in-vitro sensitive to basiliximab, an antibody able to target the Il2RA, but in-vivo investigations are needed to better assess the effects of this therapeutic approach in ALL context. Conclusions:We concluded that CD25 expression is elevated in patients with B-ALL. Our results also demonstrate that CD25 silencing induces cell cycle arrest and apoptosis. The latter result has important implications from a therapeutic point of view. Targeting CD25 receptor with anti-CD25 antibodies or peptide mimetics could be an effective strategy for targeting leukemic cells. Additionally, high CD25 expression could be exploited for the development of CAR-T therapy Disclosures Saglio: Roche:Research Funding;Pfizer:Research Funding;Incyte:Research Funding;Novartis:Research Funding;Ariad:Research Funding;Bristol-Myers Squibb:Research Funding.

Aurora B kinase as a therapeutic target in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is curable with standardized chemotherapy. However, the development of novel therapies is still required, especially for patients with relapsed or refractory disease. By utilizing an in vitro drug screening system, active molecular targeting agents against ALL were explored in this study. By the in vitro drug sensitivity test, 81 agents with various actions were screened for their cytotoxicity in a panel of 22 ALL cell lines and ALL clinical samples. The drug effect score (DES) was calculated from the dose-response of each drug for comparison among drugs or samples. Normal peripheral blood mononuclear cells were also applied onto the drug screening to provide the reference control values. The drug combination effect was screened based on the Bliss independent model, and validated by the improved isobologram method. On sensitivity screening in a cell line panel, barasertib-HQPA which is an active metabolite of barasertib, an aurora B kinase inhibitor, alisertib, an aurora A kinase inhibitor, and YM155, a survivin inhibitor, were effective against the broadest range of ALL cells. The DES of barasertib-HQPA was significantly higher in ALL clinical samples compared to the reference value. There were significant correlations in DES between barasertib-HQPA and vincristine or docetaxel. In the drug combination assay, barasertib-HQPA and eribulin showed additive to synergistic effects. Aurora B kinase was identified to be an active therapeutic target in a broad range of ALL cells. Combination therapy of barasertib and a microtubule-targeting drug is of clinical interest.

LeukmiR: a database for miRNAs and their targets in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the most common hematological malignancies in children. Recent studies suggest the involvement of multiple microRNAs in the tumorigenesis of various leukemias. However, until now, no comprehensive database exists for miRNAs and their cognate target genes involved specifically in ALL. Therefore, we developed ‘LeukmiR’ a dynamic database comprising in silico predicted microRNAs, and experimentally validated miRNAs along with the target genes they regulate in mouse and human. LeukmiR is a user-friendly platform with search strings for ALL-associated microRNAs, their sequences, description of target genes, their location on the chromosomes and the corresponding deregulated signaling pathways. For the user query, different search modules exist where either quick search can be carried out using any fuzzy term or by providing exact terms in specific modules. All entries for both human and mouse genomes can be retrieved through multiple options such as miRNA ID, their accession number, sequence, target genes, Ensemble-ID or Entrez-ID. User can also access miRNA: mRNA interaction networks in different signaling pathways, the genomic location of the targeted regions such as 3′UTR, 5′UTR and exons with their gene ontology and disease ontology information in both human and mouse systems. Herein, we also report 51 novel microRNAs which are not described earlier for ALL. Thus, LeukmiR database will be a valuable source of information for researchers to understand and investigate miRNAs and their targets with diagnostic and therapeutic potential in ALL.Database URL: http://tdb.ccmb.res.in/LeukmiR/

Abstract B134: IMPDH1 and IMPDH2 as therapeutic targets in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) tumors are dependent on purine biosynthesis for proliferation and survival and relapsed ALL is particularly associated with transcriptional up-regulation of this pathway. Increased dependency on purine biosynthesis highlights IMPDH (inosine monophosphate dehydrogenase), the rate-limiting enzyme in guanosine production, as a potential therapeutic target for the treatment of ALL. To assess the role of IMPDH1 and IMPDH2 in ALL development and progression, we generated NOTCH1-induced leukemias from Impdh1 knockout and Impdh2 conditional knockout mice. In addition, we have completed whole exome sequencing and RNAseq profiling of 12 pairs of ALL xenografts corresponding to matched diagnostic and relapsed ALL samples and have performed dose response assays with the IMPDH inhibitor mizoribine. Additionally and towards the identification of synthetic lethal genetic vulnerabilities with IMPDH inhibition in ALL we have performed a genome wide CRISPR screen in REH ALL cells with the IMPDH inhibitor mizoribine. Overall, these studies highlight the therapeutic potential for IMPDH inhibitors for the treatment of ALL. Citation Format: Chelsea Dieck, Koichi Oshima, Alberto Ambesi-Impiombato, Adolfo Ferrando. IMPDH1 and IMPDH2 as therapeutic targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B134. doi:10.1158/1535-7163.TARG-19-B134

Abstract 3695: A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common pediatric malignancy and 15-20% of patients experience disease relapse, which is frequently more aggressive and treatment resistant than the primary disease, and often has unfavorable outcome. Relapse occurs because conventional chemotherapies are unable to reliably and completely eliminate leukemia stem cells (LSCs), which are the only cells within the leukemia with the ability to self-renew and remake or replenish a leukemia from a single cell. Eliminating LSCs can greatly improve patient outcome; for example, differentiation therapy, which blocks LSC self-renewal, has improved the survival of Acute Pro-myelocytic Leukemia to >95%. We have previously completed a >8,000 animal screen in a zebrafish Myc-induced ALL model and identified a panel of zebrafish ALL, in which the leukemia stem cell frequency is ~1 in every 10 cells, and identified a unique leukemia stem cell signature. The Wnt pathway was highly expressed by LSCs, and has also emerged in mouse studies as having an important role in LSC self-renewal in T-ALL. Current Wnt inhibitors have unacceptably toxicity in the clinic. I have found that Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL3) is highly expressed by ALL cells that also express Wnt pathways genes, and is not expressed by normal cell types. In a zebrafish Myc-induce ALL model, PRL3 expression significantly enhanced leukemia stem cell frequency, while inhibition of PRL3 reduced LSC numbers in vivo. In human cells, I found that PRL3 regulates the expression of downstream Wnt pathway target genes, and we are currently testing the effects of small molecule inhibition of PRL3 on the phosphorylation status of proteins involved in Wnt signaling, to define the mechanism of PRL3 action in this pathway. Apart from identifying novel interacting partners in the Wnt pathway, I have also developed a novel zebrafish reporter line that can be used to identify other small molecules that block Wnt signaling. In these fish, Wnt expressing leukemia stem cells are fluorescently tagged. These leukemias will be used for in vivo drug screens to identify FDA-approved compounds that block Wnt signaling and/or target leukemia stem cells without adverse effects on developing zebrafish larvae. In total, my research will define a novel role for the phosphatase PRL3 in self-renewal of leukemia stem cells via activation of Wnt signaling. These data will demonstrate that “untargetable” oncogenes like Wnt can be targeted by inhibiting easily druggable critical interacting proteins, such as phosphatases like PRL3. This work is also likely to have a positive translational impact by identifying FDA-approved drugs that block LSC self-renewal in ALL and other types of Wnt and/or PRL3 dependent cancers. Citation Format: Meghan G. Haney, Kristin O'Leary, Jessica S. Blackburn. A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3695.

Abstract 2368: The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers that is diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high-dose cytotoxic chemotherapies that ALL patients are given have long-term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large-scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. Upregulation of PRL3 is correlated progression of colorectal, breast, and gastric cancers, and is associated with poor patient prognosis. We have found that PRL3 is highly expressed in a subset of patient ALL samples, and I am examining the role of PRL3 in ALL progression. We have found that PRL3 expression enhances ALL proliferation, and promotes leukemia survival after cytotoxic chemotherapy treatment. Small-molecule inhibition of PRL3 induces apoptosis in ALL, and synergizes with common ALL chemotherapies. I am currently examining the localization of PRL3 and identifying signaling pathways that it may be involved with to promote ALL progression. If my project is successful, it will provide scientific data that support our hypothesis that PRL3 is related to acute lymphoblastic leukemia. If there is a correlation between PRL3 and ALL, PRL3 could be used as a more effective and less toxic treatment for patients with ALL. Citation Format: Anna K. O'Leary, Min Wei, Jessica Blackburn. The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2368.

Human asparagine synthetase associates with the mitotic spindle.

ABSTRACTCancer cells are characterized by extensive reprogramming of metabolic pathways in order to promote cell division and survival. However, the growth promotion effects of metabolic reprogramming can be due to moonlighting functions of metabolic enzymes as well as the redirection of flux through particular pathways. To identify metabolic enzymes that might have potential moonlighting functions in oncogenesis, we have examined recent screens of the yeast GFP strain collection for metabolic enzymes that have been implicated in cancer metabolism with an unusual subcellular localization. Asparagine synthetase forms filaments in yeast in response to nutrient limitation and is part of a pathway that is a chemotherapy target in acute lymphoblastic leukemia. Interestingly, while yeast asparagine synthetase forms cytoplasmic filaments in response to nutrient stress, human asparagine synthetase is associated with the centrosomes and mitotic spindles. This localization is disrupted by both nocodazole and asparaginase treatments. This failure to localize occurs even though asparagine synthetase is highly upregulated in response to asparaginase treatment. Together, these results argue that human asparagine synthetase undergoes regulated recruitment to the mitotic spindles and that it may have acquired a second role in mitosis similar to other metabolic enzymes that contribute to metabolic reprogramming in cancer cells.

Plant homeodomain finger protein 2 as a novel IKAROS target in acute lymphoblastic leukemia.

Clinical significance of plant homeodomain finger 2 (PHF2) expressions is explored in acute lymphoblastic leukemia (ALL) patients. mRNA level was examined by qPCR. The retroviral gene expression, shRNA knockdown and chromatin-immunoprecipitation are used to observe IKAROS regulation on PHF2 transcription. PHF2 expression is significantly reduced in subsets of ALL patients, and PHF2 low expression correlates with leukemia cell proliferation and an elevation of several poor prognostic markers in B-cell ALL. IKAROS directly promotes PHF2 expression and patients with IKAROS deletion have significantly lower PHF2 expression. Casein kinase II (CK2) inhibitor significantly promotes PHF2 expression in an IKAROS-dependent manner, and casein kinase II inhibitor treatment also results in an increase of PHF2 expression and enrichment of IKAROS and H3K4me3 at PHF2 promoter in primary cells. Our results demonstrate that the IKAROS promotes PHF2 expression, and suggest that PHF2 low expression works with the IKAROS gene deletion to drive oncogenesis of ALL.

Valosin-Containing Protein/p97 as a Novel Therapeutic Target in Acute Lymphoblastic Leukemia

B acute lymphoblastic leukemia (B-ALL) cells are distinctively vulnerable to endoplasmic reticulum (ER) stress. Recently, inhibition of p97 was shown to induce ER stress and subsequently cell death in solid tumors and in multiple myeloma. We investigated the role of a novel, orally available, p97 inhibitor (CB-5083; Cleave Biosciences) in B-ALL. CB-5083 induced a significant reduction in viability in 10 human B-ALL cell lines, harboring the most common fusion-genes involved in pediatric and adult B-ALL, with IC50s ranging from 0.34 to 0.76 μM. Moreover, CB-5083 significantly reduced the colony formation of OP1 and NALM6 cells. Early and strong induction of apoptosis was demonstrated in BALL1 and OP1 cells, together with a robust cleavage of PARP. CB-5083 induced ER stress, as documented through: 1) prominent expression of chaperones (GRP78, GRP94, PDI, DNAJC3, and DNAJB9); 2) increased activation of IRE1-alpha, as demonstrated by the splicing of XBP1; and 3) activation of PERK, which resulted in a significant overexpression of CHOP, and its downstream genes. CB-5083 reduced the viability also in GRP78−/−, GRP94−/−, and XBP1−/− cells, suggesting that none of these proteins alone was strictly required for CB-5083 activity. Moreover, we showed that the absence of XBP1 (XBP1−/−) increased the sensitivity to CB-5083, leading to the hypothesis that XBP1 splicing counteracts the activity of CB-5083, probably mitigating ER stress. Finally, vincristine was synergistic with CB-5083 in both BALL1 and OP1 cells. In summary, the targeting of p97 with CB-5083 is a novel promising therapeutic approach that should be further evaluated in B-ALL.

Abstract 3033: The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia

Abstract The aggressive and unpredictable behavior of acute lymphoblastic leukemia (ALL) presents a major clinical challenge in both the pediatric and adult setting. The development of new and better chemotherapies for this disease requires a detailed understanding of the genes and pathways that drive ALL malignancy. Phosphatases are emerging as new drug targets with important roles in leukemia progression. To determine the extent to which phosphatases play a role in ALL malignancy, we used CRISPR/Cas9 to knock out expression of 255 individual phosphatases in human T-cell acute lymphoblastic leukemia cell lines and screened for deleterious effects on cell viability. Top hits from this screen included the protein tyrosine phosphatase type IVa family, also known as the PRLs. PRL3 is genomically amplified in a subset of human T-ALL, and more than 50% of human ALL patients have significantly higher expression of PRL2 and/or PRL3, compared to normal lymphocytes, suggesting that these PRLs may play an important role in ALL malignancy. Transgenic Myc-induced ALL models in zebrafish showed that overexpression of PRL2 and PRL3 significantly shortened latency of primary and relapse ALL, enhanced leukemia stem cell self-renewal (PRL2) and prevented apoptosis after standard chemotherapy treatment (PRL3). Finally, a specific PRL inhibitor strongly induced apoptosis of PRL-expressing ALL cells in a dose-dependent manner, in both zebrafish models in vivo and human cell lines in vitro. We have identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL activity and are capable of killing ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Pull-down approaches are also being used to identify the substrates of PRL phosphatase activity that are critical to ALL survival and may represent new, tractable drug targets for the treatment of ALL. <!–EndFragment–> Citation Format: Jessica S. Blackburn. The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2017-3033

Emerging Roles of MTG16 in Cell-Fate Control of Hematopoietic Stem Cells and Cancer.

MTG16 (myeloid translocation gene on chromosome 16) and its related proteins, MTG8 and MTGR1, define a small family of transcriptional corepressors. These corepressors share highly conserved domain structures yet have distinct biological functions and tissue specificity. In vivo studies have shown that, of the three MTG corepressors, MTG16 is uniquely important for the regulation of hematopoietic stem/progenitor cell (HSPC) proliferation and differentiation. Apart from this physiological function, MTG16 is also involved in carcinomas and leukemias, acting as the genetic target of loss of heterozygosity (LOH) aberrations in breast cancer and recurrent translocations in leukemia. The frequent involvement of MTG16 in these disease etiologies implies an important developmental role for this transcriptional corepressor. Furthermore, mounting evidence suggests that MTG16 indirectly alters the disease course of several leukemias via its regulatory interactions with a variety of pathologic fusion proteins. For example, a recent study has shown that MTG16 can repress not only wild-type E2A-mediated transcription, but also leukemia fusion protein E2A-Pbx1-mediated transcription, suggesting that MTG16 may serve as a potential therapeutic target in acute lymphoblastic leukemia expressing the E2A-Pbx1 fusion protein. Given that leukemia stem cells share similar regulatory pathways with normal HSPCs, studies to further understand how MTG16 regulates cell proliferation and differentiation could lead to novel therapeutic approaches for leukemia treatment.

Identification of the Energy Stress Sensor AMPK As Therapeutic Target in Acute Lymphoblastic Leukemia

Background and Hypothesis: While often transformed by the same oncogenes, biological and clinical characteristics of B-cell lineage and myeloid leukemias markedly differ. For instance, BCR-ABL1 tyrosine kinase drives both chronic myeloid leukemia (CML) and B cell lineage Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). While the majority of CML patients achieve long-term disease-free survival under treatment, Ph+ ALL patients invariably relapse within months after initial remission. Here, we investigated whether the distinct characteristics of Ph+ ALL and CML have a metabolic basis and identified the metabolic sensor LKB1 and its substrate AMPK as novel therapeutic targets in pre-B ALL.Results: Metabolic measurements revealed strikingly higher AMP:ATP ratios with concomitant decreases in ATP production in patient-derived Ph+ ALL cells when compared to CML cells. These findings indicate a state of chronic energy depletion in pre-B ALL cells. Energy deficit activates the LKB1-AMPK energy sensor pathway to stimulate glucose uptake to restore ATP levels. Notably, LKB1 levels and activity of its substrate AMPKα were higher in patient-derived Ph+ ALL cells compared to CML cells. To study the consequences of inducible deletion of Lkb1, murine BCR-ABL1-driven myeloid lineage (CML) and B cell precursor (Ph+ ALL) Lkb1fl/fl leukemia cells were generated. We found that Lkb1-deletion increased glycolysis, ATP levels and proliferation in myeloid leukemia, consistent with the common notion that LKB1 is an established tumor suppressor. On the contrary, loss of Lkb1 function resulted in diminished glycolytic activity, impaired mitochondrial functions and cell death in pre-B ALL cells. C/EBPa-mediated reprogramming of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion, restoring glycolysis, energy levels and survival of B→ myeloid reprogrammed cells. To study Lkb1 early in B cell lineage in vivo, Lkb1fl/fl mice were crossed with Mb1-Cretg/+ mice. Loss of Lkb1 function strikingly eradicated early B cell progenitor cells in vivo. Reduced survival fitness upon Lkb1 deletion in pre-B ALL cells was largely rescued by metabolites that can enter the TCA cycle and thus provide ATP. Importantly, we found that inducible deletion of Lkb1 delayed onset of pre-B ALL and prolonged survival of transplant recipient mice in vivo. Similar to observations made with deletion of Lkb1 in murine BCR-ABL1 pre-B ALL cells, both loss of Ampka function and small molecule inhibition of AMPK (BML-275) resulted in cell death as well as reduced glycolytic activity and mitochondrial function in BCR-ABL1 pre-B ALL cells. Moreover, prolonged overall survival was observed in transplant recipient mice injected with Ampka-deficient pre-B ALL cells. Finally, BML-275 synergized with glucocorticoids, a central component of all main therapy regimen in pre-B ALL, in eradicating patient-derived pre-B ALL cells.Conclusions: Taken together, our findings showed that B-cell lineage leukemia, unlike myeloid leukemia, critically depend on LKB1/AMPK signaling for survival. Our findings also showed that LKB1/AMPK can be leveraged to provide a novel therapeutic avenue in pre-B ALL. DisclosuresHochhaus:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

PP2A Is Required for B Cell Survival and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Background: The Ser/Thr Protein phosphatase 2A (PP2A) attenuates activity of RAS-ERK and PI3K-AKT signaling pathways and functions as important tumor suppressor in chronic myeloid leukemia (CML) by downregulating output of oncogenic BCR-ABL1 signaling. Restoration of PP2A activity has been proposed for the treatment of CML and eradication of TKI-resistant leukemia CML-stem cells (Neviani et al., 2013). While the tumor suppressor function of PP2A was independently confirmed by multiple groups, our analyses of PP2A function during early B cell development suggested a role for PP2A to contribute to B cell survival. Therefore, we studied whether PP2A has different roles in B-lineage ALL and CML and whether these mechanistic differences are relevant for patients with B-lineage and B-lymphoid blast crisis CML.Results: Consistent with a divergent role of PP2A in B-lineage ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A but also the catalytic subunit C and was paralleled by near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of p70S6K and S6 ribosomal protein, which indicated elevated mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. In agreement with previous studies, however, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. To verify that the observed difference between B cell and myeloid disease was indeed lineage-dependent, we reprogrammed B cell lineage ALL cells into myeloid CML using inducible overexpression of CEBPα. As expected, CEBPα-driven myeloid lineage conversion prevented cell death upon deletion of PP2A, demonstrating that PP2A-function represents a B cell-specific vulnerability.We next transplanted luciferase-labeled Ppp2r1afl/fl ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles in all cases studied demonstrating that fatal leukemia arose from few subclones that had escaped Cre-mediated deletion of PP2A.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in B cell lineage Ph+ ALL but not CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted ALL cells, together with decreased anti-oxidant gene expression, increased H2AX phosphorylation and p53 expression indicated impaired balance of glycolytic flux may account for increased death of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells.The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Cre-mediated ablation of PP2A in mouse Ph+ ALL cells induced rapid cell death through excessively oxidative stress but not in myeloid CML cells. We confirmed this pro-survival role of PP2A in human Ph+ ALL-patients derived leukemia cells through both CRISPR-Cas9 mediated genetic study and PP2A inhibition by a novel small molecule LB-100 (Jie Lu et al., 2009). These findings highlight PP2A as a therapeutic target with potential relevance in Ph+ ALL and B-lymphoid blast crisis progression of CML. DisclosuresNo relevant conflicts of interest to declare.

Erk Negative Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation ofoncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation ofErk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels,we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse modelsfor pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.

Characterization of CD22 expression in acute lymphoblastic leukemia.

CD22 is a B-lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL). Properties of CD22 expression relevant to therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B-precursor (pre-B) ALL. CD22 expression was demonstrated in all subjects (n = 163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349-19,653, n = 160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349-3,624, n = 20 versus 3,853 sites/cell, range 451-19,653, n = 140; P = <0.0001) and 6 of 21 cases had sub-populations of blasts lacking CD22 expression (22%-82% CD22 +). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti-CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti-CD22 directed agents. These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy-refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22-negative blast population.