Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1026-1026
Author(s):  
Erica A. Lehotzky ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Leukemia Cells ◽  
Inhibitory Effects ◽  
Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

The Pias-Like Coactivator ZMIZ1 Drives C-MYC Induction in Collaboration with NOTCH1 in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2380-2380
Author(s):  
Margaret Decker ◽  
Choi Li ◽  
Lesley A Rakowski ◽  
Tomasz Cierpicki ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Mouse Models ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Human T Cell ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2380 Activating NOTCH1 mutations are found in 50–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. Cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. Murine insertional mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005; Berquam-Vrieze et al., Blood, 2011). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT (PIAS)-like family. It shares a zinc finger domain, the MIZ domain, with PIAS proteins. The MIZ domain mediates interactions with DNA-binding transcription factors and sumoylation. Previously, we showed that ZMIZ1 promotes T-ALL in collaboration with leukemia-associated NOTCH1 alleles in mouse models. ZMIZ1 and activated NOTCH1 were co-expressed in a subset of human patients. Genetic ZMIZ1 inhibition slowed leukemic cell growth and overcame resistance of some T-ALL cell lines to NOTCH inhibitors. ZMIZ1 may be a new clinically relevant oncogene. Here we sought to determine the downstream target genes of ZMIZ1 in leukemia. Validation of gene expression profiling data identified C-MYC and IL7RA as downstream targets of ZMIZ1. Targeting the C-MYC or IL-7 pathways using genetic and pharmacological inhibitors partly phenocopied the growth inhibitory effects we previously saw with ZMIZ1 inhibition. In order to determine whether these genes are direct or indirect targets of ZMIZ1, we generated an estrogen fusion protein, ZMIZ1-ER. ZMIZ1-ER induced C-MYC and IL7RA expression in the presence of tamoxifen, but failed to induce these genes with the addition of cycloheximide. These data suggest that C-MYC and IL-7RA are indirect targets. Like the PIAS proteins, ZMIZ1 appeared to have a broad effect on transcription to exert its functions. We next sought to elucidate the biochemical mechanism of ZMIZ1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous c-Myc expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by several fold and rescued the C-MYC dependent cell line. ZMIZ1 enhanced the ability of even weak NOTCH1 mutants to induce C-MYC, suggesting a mechanism by which ZMIZ1 may increase resistance to NOTCH inhibitors. ZMIZ1 did not influence C-MYC expression post-transcriptionally. It functioned primarily as a transcriptional activator. Although both C-MYC and IL7RA are both NOTCH1 target genes, ZMIZ1 did not directly interact with NOTCH1 or influence the expression of several other NOTCH1 target genes such Ptcra, Hes1, Dtx1, and Cd25. Thus, ZMIZ1 did not pan-activate NOTCH signaling. Based on bioinformatic analysis, we generated mutants that deleted individual domains of ZMIZ1. All mutants expressed at high levels by Western blot. Deletion of the transcriptional activation domain or the N-terminal domain (NTD) abolished the ability of ZMIZ1 to induce c-Myc and drive proliferation. Surprisingly, deletion of the PAT-like, Proline-rich, and MIZ domains or all three domains simultaneously had no effect on ZMIZ1 function. The 120-amino acid NTD has a predicted helical structure without significant sequence homology to any known domain. It is not found in ZMIZ2 or PIAS proteins. In summary, the mechanism of ZMIZ1 appears to be novel, indirect, transcriptional, and independent of canonical NOTCH and PIAS functions. Our study demonstrates the importance of characterizing genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phloridzin Docosahexaenoate (PZ-DHA) : A Novel Naturally Derived Drug Active in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5197-5197
Author(s):  
Niroshaathevi Arumuggam ◽  
Nicole Melong ◽  
Catherine K.L. Too ◽  
Jason N. Berman ◽  
H.P. Vasantha Rupasinghe
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Jurkat Cells ◽  
Interferon Α ◽  
Omega 3 ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease that accounts for about 15% of pediatric and 25% of adult ALL. Although risk stratification has provided more tailored therapy and improved the overall survival of T-ALL patients, clinical challenges such as suboptimal drug responses, morbidity from drug toxicities, and drug resistance still exist. Plant polyphenols have therapeutic efficacy as pharmacological adjuvants to help overcome these challenges. They can be acylated with fatty acids to overcome issues concerning bioavailability, such as poor intestinal absorption and low metabolic stability. Phloridzin (PZ), a flavonoid found in apple peels, was acylated with an omega-3 fatty acid, docosahexaenoic acid (DHA), to generate a novel ester called phloridzin docosahexaenoate (PZ-DHA). The cytotoxic effect of PZ-DHA was studied in the human Jurkat T-ALL cell line. PZ-DHA significantly reduced the viability and cellular ATP levels of treated cells. PZ-DHA was found to selectively induce apoptosis in Jurkat cells, while sparing normal murine T-cells. Apoptosis was further confirmed by demonstrating the ability of PZ-DHA to induce morphological alterations, DNA fragmentation, caspase activation, and the release of intracellular lactate dehydrogenase. PZ-DHA also significantly inhibited cell division in Jurkat cells. Furthermore, interferon-α-induced phosphorylation of the transcription factor, STAT3, was downregulated following PZ-DHA treatment. The in vitro efficacy of PZ-DHA was recapitulated in vivo in an established zebrafish xenograft model, where the proliferation of transplanted Jurkat cells was inhibited when PZ-DHA was added to the embryo water. Overall, these findings provide evidence for PZ-DHA as a novel therapeutic agent with activity in T-ALL. Studies examining the effect of PZ-DHA on patient-derived ALL cells engrafted in zebrafish are currently underway. Disclosures No relevant conflicts of interest to declare.

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2641-2641
Author(s):  
Suning Chen ◽  
Stefan Nagel ◽  
Bjoern Schneider ◽  
Maren Kaufmann ◽  
Ursula R. Kees ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Small Sample ◽  
Upstream Region ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Whole Transcriptome Sequencing Identified a Distinct Subtype of Acute Lymphoblastic Leukemia with Abnormalities of CREBBP and EP300

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3912-3912
Author(s):  
Hui Zhang ◽  
Maoxiang Qian ◽  
Shirley, Kow Yin Kham ◽  
Shuguang Liu ◽  
Chuang Jiang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Rearrangements ◽  
Ectopic Expression ◽  
Cytotoxic Agents ◽  
Hematopoietic Stem ◽  
Exon 2 ◽  
Childhood All

Abstract While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1322-1322
Author(s):  
Manabu Kusakabe ◽  
Ann Chong Sun ◽  
Kateryna Tyshchenko ◽  
Rachel Wong ◽  
Aastha Nanda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

scholarly journals Development of Proteolytic Targeting Chimeras to Target Lck in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 867-867
Author(s):  
Jianzhong Hu ◽  
Jamie Jarusiewicz ◽  
Jaeki Min ◽  
Lei Yang ◽  
Divyabharathi Chepyala ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
E3 Ligase ◽  
Proteomic Profiling ◽  
Drug Removal ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Lck Protein

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy for which novel therapies are much needed especially in patients with relapsed diseases. By combining large-scale ex vivo pharmacotype profiling with network-based systems biology analyses, our group recently identified LCK dependency as a therapeutic vulnerability in 44% of T-ALL in children (Nat Cancer 2, 284-299, 2021). LCK inhibitors such as dasatinib exhibit striking anti-leukemia effects in this T-ALL subset. However, the transient LCK inhibition by dasatinib only resulted in incomplete response to monotherapy unless the drug was delivered continuously at a high level. Therefore, it is imperative to develop novel agents that produce sustained suppression of LCK signaling in T-ALL. To this end, we synthesized a set of proteolytic targeting chimeras (PROTACs) that target LCK and cereblon (CRBN) E3 ligase. These PROTACs bind and recruit LCK to CRBN E3 ligase, rendering LCK susceptible to ubiquitination and ultimately proteasomal degradation. Cell viability assay was performed in an LCK-dependent T-ALL cell line KOPT-K1 to determine its sensitivity to this panel of PROTACs. PROTACs showed up to 6.9-fold improvement in cytotoxicity relative to dasatinib. To validate PROTAC-induced LCK degradation, KOPT-K1 cells were treated with PROTACs or dasatinib at 100 nM for 24 hours and LCK protein was quantified by western blotting. LCK degradation occurred rapidly in the presence of PROTAC agents whereas dasatinib treatment did not affect LCK abundance. Furthermore, PROTAC-induced apoptosis of T-ALL cells was abolished by lenalidomide, a CRBN binder, suggesting that their cytotoxic effects were mediated by CRBN-driven LCK degradation. We also validated these findings in patient-derived T-ALL samples. In addition, we determined solubility, permeability, and stability of these PROTACs in vitro. Based on anti-leukemia effects as well as physical chemical properties, we prioritized PROTACs SJ001011646 and SJ001011447 as our top candidates for further evaluations. We hypothesized that the catalytic protein degradation by PROTACs will produce a more sustained suppression of the LCK signaling compared to transient LCK inhibition by dasatinib. To test this, we performed a wash-out assay comparing anti-leukemic effects of top PROTACs with dasatinib. KOPT-K1 cells were treated with vehicle, dasatinib, SJ001011646 and SJ001011447, respectively at 100 nM for 18 hours. Cells were then washed and placed in drug-free culture with viability monitored daily. The vehicle treated cells exhibited an exponential growth while drug treated groups showed dramatic growth inhibition within 2 days. Notably, dasatinib-treated cells continued to undergo apoptosis for 96 hours after drug removal before started to recover. By contrast, SJ001011447 treatment repressed cell growth for 144 hours post wash-out. Most impressively, there was no evidence of growth recovery in cells treated SJ001011646 even 240 hours after drug removal. In addition, we confirmed the formation of ternary complex of LCK, PROTAC, and E3 ligase, using the AlphaLISA assay. To systematically identify therapeutic targets of PROTACs, we performed proteomic profiling of KOPT-K1 cells before and after drug treatment in vitro. Cells were treated with vehicle, SJ001011447 or SJ001011646 at 100 nM for 24 hours before harvested for Tandem Mass Tag-based proteomic profiling. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which 34 and 35 were significantly changed by SJ001011447 and SJ001011646, respectively (p&lt;0.05, foldchange &gt;2 or &lt;-2). LCK was among proteins most significantly reduced after PROTAC treatment. Finally, we also developed formulation for PROTAC SJ001011646 for in vivo testing; and preclinical pharmacokinetic and pharmacodynamic characterization of this molecule is ongoing using T-ALL xenograft models. In conclusion, we developed LCK-targeting PROTACs with potent anti-leukemia effects. Highly effective in degrading LCK protein, these agents produced sustained LCK suppression superior to small molecule inhibitors, pointing to novel strategies to therapeutically target LCK in T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals MiR-7 Functions as a Tumor Suppressor by Targeting the Oncogenes TAL1 in T-Cell Acute Lymphoblastic Leukemia

2020 ◽  
Vol 19 ◽  
pp. 153303382093413
Author(s):  
Hongbo Sun ◽  
Zhifu Zhang ◽  
Wei Luo ◽  
Junmin Liu ◽  
Ye Lou ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Lymphoblastic Leukemia ◽  
Critical Role ◽  
Ectopic Expression ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Acute Lymphoblastic Leukemia ◽  
And Migration

Background: T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis. Material and Method: To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression. Results: We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1. Conclusions: These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.

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