Mature T-cell Acute Lymphoblastic Leukemia Research Articles

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

ALL-220 Azacitidine in Combination With Venetoclax Maintenance Post Allogeneic Hematopoietic Cell Transplantation in T-Cell Acute Lymphoblastic Leukemia

Relapsed and/or refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few available salvage options and usually carries a dismal outcome with only 10% of patients surviving at 5 years. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy in R/R T-ALL remains problematic, as up to half of patients will relapse post allo-HSCT. Post-transplant maintenance in acute myeloid leukemia (AML) has long been debated, with recent implementation by transplant centers, especially the use of hypomethylating agents (HMAs) and targeted agents such as FLT3 inhibitors. In T-ALL, this concept has not been explored yet with very little data. The aim of this case series is to present a novel approach for post-transplant maintenance in patients diagnosed with R/R T-ALL. This is a case series including 4 patients with R/R T-ALL who underwent allo-HSCT at the American University of Beirut Medical Center and received post-transplant maintenance with low-dose 5-azacitidine and venetoclax. We identified four adult patients diagnosed with high-risk T-ALL (three patients with mature T-ALL and one with early T-precursor [ETP] ALL) with a median age of 35 years (range, 31-45 years). All received post-allo-HSCT maintenance with 5 days of low dose 5-azacitidine at 32 mg/m2 daily with venetoclax 100 mg orally daily for 5 days with voriconazole every 28 days. The median duration of maintenance treatment was 12.75 months (range, 9.5-23.5 months). All patients remain in continuous complete remission with undetectable measurable residual disease after a median follow-up of 15 months from the date of allo-HSCT (range, 12-25 months). Both HMAs and venetoclax possess significant antitumor activity effects against AML/myelodysplastic syndrome, and their efficacy in R/R T-ALL has been demonstrated in multiple case series. These encouraging results along with our case series of successful maintenance treatment with azacitidine with venetoclax in T-ALL are worth exploring in larger prospective trials.

CRLF2 overexpression defines an immature-like subgroup which is rescued through restoration of the PRC2 function in T-cell precursor acute lymphoblastic leukemia.

CRLF2 overexpression has been described as a biomarker of poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). In the present study, we aimed to unravel the genomic profile underlying CRLF2 overexpression (CRLF2-high) by analyzing RNA-seq, WES, and SNP-array data from 264 T-ALL patients and five cell lines deposited on the TARGET initiative, Cancer Cell Line Encyclopedia and Gene Expression Omnibus. These data allowed us to delineate the genomic landscape of CRLF2-high in T-ALL, which was associated with PTEN, JAK3, PHF6, EZH2, and RUNX1 mutations. We also observed an enrichment of CRLF2-high in early T-precursor (ETP)-ALL (23.08% vs. 4.02%, P=7.579e-06 ) and a very similar gene upregulation profile between these two entities. The inhibition of BET (iBET) proteins is a strategy previously demonstrated to reverse the gene upregulation pattern of ETP cells through restoration of polycomb repressive complex 2 (PRC2) activity. While CRLF2 expression was rescued by using this strategy in LOUCY (untreated vs. iBET P =0.0095, DMSO vs. iBET P=0.0286), a classical ETP-ALL cell line, PRC2 loss was not sufficient to promote CRLF2 upregulation in JURKAT, a more mature T-ALL cell line. Considering the role of IKZF1 in CRLF2 regulation and in recruitment of PCR2, we evaluated IKZF1 status according to CRLF2-expression subgroups. We identified that IKZF1 transcripts with intron retention were upregulated in the CRLF2-high subgroup. Here, we delineated the gene expression profile of CRLF2-high T-ALL samples and unraveled the crucial role of PRC2 in CRLF2 regulation in ETP-ALL.

Intracellular Cholesterol Pools Regulate Oncogenic Signaling and Epigenetic Circuitries in Early T-cell Precursor Acute Lymphoblastic Leukemia.

Overtly distinct cell metabolic pathways operate in ETP- and T-ALL pointing to specific metabolic vulnerabilities. Inhibition of mevalonate biosynthesis selectively blocks oncogenic AKT-MYC signaling in ETP-ALL and suppresses leukemia cell growth. Ultimately, these results will inform the development of novel tailored and more effective treatments for patients with high-risk ETP-ALL. This article is highlighted in the In This Issue feature, p. 587.

Calcium Signaling Is Impaired in PTEN-Deficient T Cell Acute Lymphoblastic Leukemia.

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.

MAP3K7 Is Recurrently Deleted in Pediatric T-Lymphoblastic Leukemia and Affects Cell Proliferation Independently of NF-κB

Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which is crucial for innate immune signaling. It is functionally and prognostically relevant both in hematologic malignancies and solid tumors. However, its distinct biological functions and relevance for different tumor entities are cell-type specific and remain controversial. Here, we show that MAP3K7 is recurrently deleted in pediatric T-ALL cases and that a residual expression of MAP3K7 is indispensable for T-ALL cell lines.We determined the frequency of MAP3K7 deletions in a cohort of 327 pediatric T-ALL patients by multiplex ligation-dependent probe amplification (MLPA). In a subgroup, we additionally employed targeted sequencing to identify point mutations. Deletions of MAP3K7 were identified in approximately 10% and point mutations in approximately 1% of children with T-ALL. All deletions of 6q15 were heterozygous and the adjacent CASP8AP2 gene was co-deleted in 32 of 33 cases. Clinically, the MAP3K7/CASP8AP2 deletions were significantly associated with a mature T-ALL immunophenotype, but not with any other patient characteristics or with the cumulative incidence of relapse and overall survival. Furthermore, deletions of MAP3K7/CASP8AP2 were preferentially associated with SIL-TAL1 fusions. Within the subgroup of SIL-TAL1 positive patients, those with MAP3K7/CASP8AP2 deletion showed a trend towards a higher risk of relapse compared to SIL-TAL1 -positive MAP3K7/CASP8AP2 -negative patients (25% vs 8%, p=0.13).We next analyzed the effects of shRNA-mediated MAP3K7 depletion on cell proliferation, apoptosis and NF-κB activation in three T-ALL cell lines (CCRF-CEM, Jurkat, MOLT4) that do not carry MAP3K7 deletions. We aimed at phenocopying a MAP3K7 deletion by using an Adeno-associated viral vector-mediated transduction system to efficiently deliver anti-MAP3K7 shRNA together with a GFP reporter to these cell lines. Transduction efficiency was above 80% for the cell lines CCRF-CEM and Jurkat. Depletion efficiency was uniformly in the range of 75%. Treatment with anti-MAP3K7 shRNA in comparison to non-silencing shRNA significantly slowed down proliferation in all three T-ALL cell lines and resulted in an increase of apoptotic cells up to 8-fold as measured by Annexin V staining.In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Stimulation with TNF-α led to rapid and transient degradation of IκB and an accumulation of phospho-NF-κB p65 (Ser536), while other NF-κB proteins (p100, p105, p50) remained unaffected. Depletion of MAP3K7 neither changed IκB levels in unstimulated cells nor did it prevent the degradation of IκB after stimulation with TNF-α. Furthermore mRNA expression of none of the NF-κB target genes was consistently changed by more than 2-fold in any of the cell lines after MAP3K7 depletion. We conclude that in T-ALL cell lines, MAP3K7 is not required for the degradation of IκB and subsequent activation of NF-κB after stimulation with TNF-α.This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts. DisclosuresSchrappe:Medac: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

T-Cell Acute Lymphoblastic Leukemia Can be Subdivided into Six Genetically Distinct Subtypes with Prognostic Impact By Combination of Whole Genome and Whole Transcriptome Data

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a rare aggressive neoplasm accounting for ∼20% of all ALL cases. It is more common in adults than in children, although the incidence decreases with older age. Subclassification of T-ALL cases according to WHO is so far solely based on the immunophenotype. Although a number of common molecular aberrations have been described in T-ALL, a molecular classification of T-ALLs so far is missing. Aim: (1) Molecular subclassification of T-ALL cases based on whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) data. (2) Analysis of correlations between aberrations, subgroups and other parameters. Methods: WGS and WTS were performed in 114 patients. For WGS, 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). For WTS, 101 bp paired-end reads were produced on a NovaSeq 6000 system with a yield between 35 and 125 million paired reads per sample. SPSS (version 19.0.0, IBM Corporation, Armonk, NY) was used for statistical analysis. Results: The cohort comprised 114 T-ALL cases (29% female, 71% male) with a median age of 37 years (range 1 - 91 years). Based on mutations (mut), translocations and gene expression, the cases were subdivided into six different groups: group 1 (n=20) was defined by overexpression (oex) of TLX1 (by t(10;14)(q24;q11)/TRAD-TLX1, n=17; t(7;10)(q34;q24)/TRB-TLX1, n=2; inv(10)(q23q24), n=1) and was correlated to a high frequency of mut in NOTCH1 (19/20, 95%, p=0.011 compared to the other T-ALL cases), PHF6 (11/20, 55%, p=0.04) and BCL11B (5/20, 25%, p=0.004). Group 2 (n=9) showed TLX3 oex by t(5;14)(q35;q32)/BCL11B-TLX3 (n=8) or t(5;8)(q35;q24) (n=1). Mut in WT1 (5/9 = 56%, p<0.001) and PHF6 (7/9 = 78%, p=0.005) were frequently detected. Groups 1/2 were further characterized by male predominance (18/20 = 90%, p=0.04 and 8/9 = 89%) and a cortical immunophenotype (20/20 = 100% and 7/9 = 78%). Group 3 (n=23) was defined by oex of HOXA genes, caused by inv(7)(p15q34)/TRB-HOXA (n=6), other HOXA rearrangements (n=2), SET-NUP214 (n=6), NUP98-RAP1GDS1 (n=3) and fusions involving MLLT10 (n=6). Group 4 (n=7) was defined by oex of TAL1 due to the fusion STIL-TAL1 (n=3), t(1;14)(p33;q11)/TRA-TAL1 (n=1), mut in MYB enhancers (n=2) and a MYB-translocation (n=1), and was correlated to the absence of PHF6 mut (p=0.045). The remaining 55 cases did not show oex of TLX1, TLX3, HOXA or TAL1 and were subdivided based on the presence of NOTCH1 aberrations (group 5, n=29, NOTCH1+; group 6, n=26, NOTCH1-). 25/29 NOTCH1+ cases showed at least 1 mut in NOTCH1 (range: 1-3), 2/29 had a t(7;9)(q34;q34)/TRB-NOTCH1, 2/29 showed NOTCH1 deletions. Group 5 correlated to mut in DNMT3A, NRAS and ASXL1 (p=0.001; p=0.006; p<0.001) and comprised a high number of female patients (15/29 = 52%, p=0.004). NOTCH1- cases lacked mut in FBXW7 and DNM2 (p=0.02) and comprised a high number of cases with Pre-T-ALL and mature T-ALL immunophenotype. Other findings include: (1) detection of two cases with a novel type of NOTCH1 mut, termed NOTCH1-ITD, characterized by a duplication event in the NOTCH domain. (2) Mut in DNMT3A, TET2 and ASXL1 were exclusively detected in groups 5 and 6, causing correlation to older age (p=0.011 and p=0.001), whereas the HOXA group was correlated to younger age (p=0.001) (3) NRAS mut were associated with females (6/10 = 60% vs. 27/104 = 26%, p=0.023). (4) Oex of BCL11B and RAG1 was predominantly detected in groups 1 and 4. (5) Mut in DNM2 occurred concomitantly with NOTCH1 mut (p<0.001); NOTCH1 and PHF6 mut were correlated with each other (p=0.008). (6) Female patients were significantly older than male patients (55 vs. 36 years, p=0.005). (7) Finally, analysis of OS (total cohort median: 36 months (m)) in the six groups revealed a significantly more favorable outcome of groups 1 (TLX1; n.r.) and 3 (HOXA; n.r.) compared to the other groups (see table), which might be linked to the high number of cases with cortical immunophenotype. Conclusions: (1) The combination of WGS and WTS successfully subclassified T-ALL cases on a molecular level into six different groups, by oex of TLX1, TLX3, HOXA or TAL1 and presence/absence of NOTCH1 mut. (2) We identified a novel so far unknown type of NOTCH1 aberrations, termed NOTCH1-ITD. (3) Molecular sub-classification of T-ALL cases also impacts on prognosis (TLX1 and HOXA group, longer OS; NOTCH1+ and TAL1 group, shorter OS). Figure Disclosures No relevant conflicts of interest to declare.

Characterization of T-ALL-Specific Heterogenous Blast Populations Using High Resolution Single Cell Profiling

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is characterized by proliferation of immature T-cells and accounts for ~15% of pediatric ALL. T-ALL blasts are phenotypically diverse and are sub-classified into pro-, pre-, cortical and mature T-ALL based on the stage of differentiation of the leukemic clone. Early T precursor ALL (ETP-ALL) is a T-ALL subtype associated with higher risk of relapse (Raetz and Teachey, 2016). Bulk sequencing approaches have revealed valuable information about modulated genes in T-ALL; however, little is understood about the interplay between tumor cells and the immune microenvironment. We present a comprehensive single cell RNA sequencing (scRNASeq) analysis of T-ALL samples with the purpose of characterizing the heterogenous tumor and microenvironment cells in order to identify dysregulated genes in leukemia cells and investigate oncogenic signaling pathways. Methods: We profiled 16,280 cells from 5 diagnostic pediatric T-ALL bone marrow samples using the Chromium single cell transcriptomics platform (10x Genomics, Pleasanton, CA). To compare T-ALL versus healthy bone marrow profiles and identify T-ALL-specific malignant blast cell populations, we included data from 3 healthy pediatric bone marrow samples from a recent study (Caron et al, 2020). Dimension reduction using the Uniform Manifold Approximation and Projection (UMAP) approach was used to identify unique cell type clusters (Becht et al, 2018). Using the TARGET dataset (https://ocg.cancer.gov/programs/target), we further evaluated the prognostic significance of identified malignant blast-specific genes by performing Kaplan-Meier survival analysis and compared gene expression patterns and tumor microenvironment makeup between ETP-ALL and non-ETP T-ALL patient samples. Results: We successfully characterized leukemic blasts (CD7+, CD99+ and CD3D+) and other major immune cell types (T cells, B cells, monocytes, erythroid precursors) using the expression of established marker genes (Fig 1A, C). Clustering analysis revealed patient-specific leukemia blast cell clusters (Fig 1B). Differential expression analysis between CD3D+ patient-specific leukemia clusters and CD3D+ clusters comprised of normal T-cells identified a set of 385 promiscuous genes that are significantly differentially expressed between malignant and normal T-cells (p-value <0.05) despite tumor cell heterogeneity. Among the top genes upregulated in the leukemia clusters are HES4 (a downstream target of NOTCH1), CD99, RACK1, and TUBB (Fig 1D), which have previously been implicated in T-ALL leukemogenesis and chemoresistance (DeDecker et al, 2020; Cox et al, 2016; Lei et all 2016). Further, pathways analysis demonstrated significant activation (Z score >1.3, p-value <0.05) of multiple pathways associated with cancer stemness, cellular growth and proliferation including PI3K/AKT, unfolded protein response, and glycolysis, implicating these genes as oncogenic mediators in T-ALL blasts (Fig 2A). Overexpression of S100A4, IFITM2, and CD74 were significantly associated with poor survival in the TARGET cohort of 241 patients (hazard ratio 2.2, 5.8, and 7.1, respectively, p-value <0.05), indicating their prognostic significance (Fig 2B). We additionally evaluated the expression of our gene set across ETP-ALL and non-ETP T-ALL groups in the TARGET dataset. Multiple leukemic blast-specific genes including ARMH1, CD44, CD74, DNAJC1, and IFITM2 were significantly upregulated in ETP-ALL. Further, deconvolution analysis performed on ETP-ALL vs non-ETP T-ALL samples determined that tumor microenvironment cell types are differentially enriched in these T-ALL groups. We observed enrichment of memory B cells, dendritic cells, NK cells, and M2 macrophages in ETP-ALL samples as compared to non-ETP T-ALL samples. Conversely,lower levels of mast cells and T-regulatory cells were observed in ETP-ALL samples. Conclusions: We identified a gene signature characterizing heterogenous T-ALL blast populations. External validation using the TARGET dataset identified genes within the signature that are associated with poor outcomes in T-ALL. Differences in the composition of the immune microenvironment in ETP-ALL versus non-ETP T-ALL samples provide a promising area for future study. Further studies will be carried out with relapsed and non-relapsed T-ALL samples to validate this leukemia signature. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

PIM Kinase Inhibitors Block the Growth of Primary T-cell Acute Lymphoblastic Leukemia: Resistance Pathways Identified by Network Modeling Analysis.

Despite significant progress in understanding the genetic landscape of T-cell acute lymphoblastic leukemia (T-ALL), the discovery of novel therapeutic targets has been difficult. Our results demonstrate that the levels of PIM1 protein kinase is elevated in early T-cell precursor ALL (ETP-ALL) but not in mature T-ALL primary samples. Small-molecule PIM inhibitor (PIMi) treatment decreases leukemia burden in ETP-ALL. However, treatment of animals carrying ETP-ALL with PIMi was not curative. To model other pathways that could be targeted to complement PIMi activity, HSB-2 cells, previously characterized as a PIMi-sensitive T-ALL cell line, were grown in increasing doses of PIMi. Gene set enrichment analysis of RNA sequencing data and functional enrichment of network modules demonstrated that the HOXA9, mTOR, MYC, NFκB, and PI3K-AKT pathways were activated in HSB-2 cells after long-term PIM inhibition. Reverse phase protein array-based pathway activation mapping demonstrated alterations in the mTOR, PI3K-AKT, and NFκB pathways, as well. PIMi-tolerant HSB-2 cells contained phosphorylated RelA-S536 consistent with activation of the NFκB pathway. The combination of NFκB and PIMis markedly reduced the proliferation in PIMi-resistant leukemic cells showing that this pathway plays an important role in driving the growth of T-ALL. Together these results demonstrate key pathways that are activated when HSB-2 cell line develop resistance to PIMi and suggest pathways that can be rationally targeted in combination with PIM kinases to inhibit T-ALL growth.

The Profile of Immunophenotype and Genotype Aberrations in Subsets of Pediatric T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous malignancy, which reflects distinctive stages of T-cell differentiation arrest. We have revisited a cohort of pediatric T-ALL, in order to test if immunophenotypes associated with molecular alterations would predict the patient's outcome. Genetic mutations, translocations and copy number alterations were identified through Sanger sequencing, RT-PCR, FISH and multiplex ligation-dependent probe amplification (MLPA). We defined 8 immunophenotypic T-ALL subtypes through multiparametric flow cytometry: early T-cell precursor (ETP, n = 27), immature (n = 38), early cortical (n = 15), cortical (n = 50), late cortical (n = 53), CD4/CD8 double negative mature (n = 31), double positive mature (n = 35) and simple positive mature (n = 31) T-ALL. Deletions (del) or amplifications (amp) in at least one gene were observed in 87% of cases. The most frequent gene alterations were CDKN2A/Bdel (71.4%), NOTCH1mut (47.6%) and FBXW7mut (17%). ETP-ALL had frequent FLT3mut (22.2%) and SUZ12del (16.7%) (p < 0.001), while CDKN2A/Bdel were rarely found in this subtype (p < 0.001). The early cortical T-ALL subtype had high frequencies of NOTCH1mut and IL7Rmut (71%, 28.6%, respectively), whereas, mature T-ALL with double positive CD4/CD8 had the highest frequencies of STIL-TAL1 (36.7%), LEF1del (27.3%) and CASP8AP2del (22.7%). The co-existence of two groups of T-ALL with NOTCH1mut/IL7Rmut, and with TLX3/SUZ12del/NF1del/IL7Rmut, were characterized with statistical significance (p < 0.05) but only STIL-TAL1 (pOS 47.5%) and NOTCH1WT/FBXW7WT (pOS 55.3%) are predictors of poor T-ALL outcomes. In conclusion, we have observed that 8 T-ALL subgroups are characterized by distinct molecular profiles. The mutations in NOTCH1/FBXW7 and STIL-TAL1 rearrangement had a prognostic impact, independent of immunophenotype.

Abstract 5820: Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

Abstract While intensive chemotherapy regimens result in remission in approximately 80% of pediatric and 45% of adult ALL patients, new approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Early T-cell precursor (ETP) ALL, a subset of T-ALL associated with a higher rate of relapse and induction failure, with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. PIM protein kinases are overexpressed in a number of hematopoietic malignancies, including ALL, with its overexpression being associated with poor prognosis. Pan-PIM inhibitors (PIMi) are being tested in clinical trials in patients with relapsed/refractory multiple myeloma and acute myeloid leukemia. Analysis of the effects of PIMi (AZD1208/LGB321) on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Immunophenotype of all the PIMi insensitive cells lines was consistent with a more mature T-ALL phenotype. In contrast, sensitive cells, had an immature or ETP-ALL phenotype. Bioinformatics analysis of three independent T-ALL patient datasets (GSE28703, E-MEXP-313, and GSE62156) demonstrated overexpression of PIM1 in the majority of ETP-ALLs and in a small subset of non-ETP-ALL. We also identified 58 genes changing significantly between PIMi sensitive versus insensitive cell lines that were common to the differential genes between ETP versus non-ETP ALL cases from GSE28703, suggesting that high PIM1 expression along with 58 gene signature could be a driver of the ETP phenotype. Importantly, we have made the novel observation that, combination of Ponatinib, a tyrosine kinase inhibitor (TKI) with the PIMi is synergistically lethal to T-ALL cells with ETP phenotype. These agents appear to work together by inducing PARP and Caspase cleavage, sufficient to drive apoptosis. Following engraftment of NSG mice with H-SB2-Luc cells, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly abrogated leukemia as evidenced by optical scanning for luciferase cells, and reduced number of hCD45+ leukemic cells in the peripheral blood and bone marrow. Also, the dual therapy significantly prolonged the survival of the treated mice. In summary, our work demonstrates a strong preclinical rationale for a novel treatment strategy of combining PIMi and TKI to treat T-ALL patients with high PIM expression. Citation Format: Sathish Kumar Reddy Padi, Libia Luevano, Neha Singh, Jiin Song, Ritu Pandey, Jon C. Aster, Xue-Zhong Yu, Shikhar Mehrotra, Andrew Kraft. Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset [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 5820. doi:10.1158/1538-7445.AM2017-5820

Therapeutic Targeting of PIM Protein Kinases in a Subset of T-Cell Acute Lymphoblastic Leukemia

New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies.To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156.Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 < 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (>9 folds, p<0.01), activated JAK/STAT pathway and lower levels of MYC. Sensitive cells (H-SB2 and DU.528), had an immature or ETP-ALL phenotype (CD4-/CD8- and TdT-). Insensitive CUTLL1, SUP-T1, and HPB-ALL (XTT assay: IC50 > 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells.While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI < 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rγ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p<0.05), and reduced the number of leukemic cells (hCD45 staining) in the peripheral blood and bone marrow. This dual therapy significantly prolonged the survival (p<0.005) of the treated mice. This outcome was obtained at one-tenth of normal dose of Ponatinib used in animals, suggesting that in the humans the side effects induced by this agent could be avoided by using this combination therapy.Taken together, these findings highlight the important role for PIM protein kinase activity in T-ALL and especially in the ETP-ALL, phenotype. Tumor xenograft experiments provide strong preclinical rationale for a novel treatment strategy of combining PIM and tyrosine kinase inhibitors for treatment of patients with PIM overexpressing T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.

Autologous hematopoietic stem cell transplantation as late high-dose consolidation in adult patients with T-cell lymphoblastic leukemias: Results of a Russian multicenter study

To analyze the efficiency of the ALL-2009 protocol (ClinicalTrials.gov NCT01 193933) in patients with T-cell leukemias, particularly the role of autologous hematopoietic stem cell transplantation (auto-HSCT) after non-myeloablative BEAM conditioning, followed by maintenance therapy. Since 2009, the ALL-2009 study has enrolled 90 patients with T-cell acute lymphoblastic leukemia (T-ALL), the treatment results were assessed in 86 patients: 6 and 28 patients underwent allogeneic HSCT and auto-HSCT, respectively. A landmark analysis was used to compare survival rates in patients who had undergone auto-HSCT and in those who had not. For this, the median time from complete remission to the date of auto-HSCT was determined (the median was 6 months). Then to compare with the auto-HSCT group, only 27 patients who had been in complete remission for 6 months or more were included in a chemotherapy group. The achievement of complete remission in patients with thymic T-ALL (100%) was significantly higher than in those with early (85.7%) or mature (70%) variants. The patients with early and mature T-ALL as compared to those with thymic T-ALL showed high death rates in the remission induction (7.4 and 10% versus 0) and the patients with mature T-ALL had a.higher proportion of refractory forms (20% versus 0). The 5-year overall and relapse-free survival rates in all the T-ALL patients were 66 and 76%, respectively. After auto-HSCT, the risk of recurrence was 0% versus 21% after chemotherapy (p=0.03). The relapse-free survival rates significantly differed in the auto-HSCT and non-auto-HSCT groups: 100 and 66%, respectively (p=0.047). The long-term survival rates obtained during this multicenter study in the T-ALL patients treated according to the ALL-2009 protocol, the basis for which is the principle of continuity of cytostatic effects, are exclusively optimistic. Late consolidation with auto-HSCT following non-myeloablative BEAM conditioning, followed by maintenance therapy, considerably reduces the risk of recurrence.

Prognostic relevance of integrated genetic profiling in adult T-cell acute lymphoblastic leukemia

AbstractAdult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor associated with poor outcome. In this study, we analyzed the prognostic relevance of genetic alterations, immunophenotypic markers, and microarray gene expression signatures in a panel of 53 adult T-ALL patients treated in the Eastern Cooperative Oncology Group E2993 clinical trial. An early immature gene expression signature, the absence of bi-allelic TCRG deletion, CD13 surface expression, heterozygous deletions of the short arm of chromosome 17, and mutations in IDH1/IDH2 and DNMT3A genes are associated with poor prognosis in this series. In contrast, expression of CD8 or CD62L, homozygous deletion of CDKN2A/CDKN2B, NOTCH1 and/or FBXW7 mutations, and mutations or deletions in the BCL11B tumor suppressor gene were associated with improved overall survival. Importantly, the prognostic relevance of CD13 expression and homozygous CDKN2A/CDKN2B deletions was restricted to cortical and mature T-ALLs. Conversely, mutations in IDH1/IDH2 and DNMT3A were specifically associated with poor outcome in early immature adult T-ALLs. This trial was registered at www.clinicaltrials.gov as #NCT00002514.

T-Cell Lymphoblastic Lymphoma and T-Cell Acute Lymphoblastic Leukemia: A Separate Entity?

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are considered the same disease, differing by the extent of bone marrow infiltration. According to recent gene expression profiling data, T-ALL and T-LBL can be separated by prediction analysis of microarrays showing an overexpression of MML1 in T-LBL and CD47 in T-ALL. Immunophenotypes of T-LBL and T-ALL are identical but differ in frequency, with a higher rate of cortical or mature immunophenotypes in T-LBL, which is probably related to the higher rate (> 90%) of mediastinal tumors. Treatment approaches in T-LBL changed from conventional non-Hodgkin lymphoma (NHL) protocols to intensive NHL protocols but recently to ALL-designed protocols. T-ALL remission rates are 90%, and overall survival (OS) has improved to 60%–70%. Mediastinal tumors resolve in most cases of T-ALL with chemotherapy only, whereas in T-LBL additional mediastinal irradiation seems to be beneficial. Strategies for stem cell transplantation (SCT) in T-LBL and T-ALL differ. Autologous SCT in complete remission (CR) in T-LBL gives a 70% survival rate, which is similar to chemotherapy alone. In T-ALL, the subtypes of early and mature T-ALL have a poor outcome with chemotherapy alone (< 30%) and might profit from an allogeneic transplantation in first CR (OS > 50%). There seems to be no need for transplantation in thymic T-ALL in first CR. Prognostic factors are published for T-ALL but not for T-LBL. MRD may guide further treatment strategies in T-ALL and probably also in T-LBL as indications for a SCT or for the evaluation of novel, particularly T-cell–specific, drugs.

CD56 expression in T-cell acute lymphoblastic leukemia is associated with non-thymic phenotype and resistance to induction therapy but no inferior survival after risk-adapted therapy

Expression of CD56 has been associated with poor prognosis in acute myeloid leukemia and aggressive lymphoma. We analyzed the impact of CD56 expression in a cohort of 452 newly diagnosed adult T-cell acute lymphoblastic leukemia (T-ALL) patients; clinical data were available for 306 patients. Treatment was according to the GMALL study protocols 06/99 and 07/03 stipulating stratification into standard (thymic T-ALL) and high risk (pre- and mature T-ALL) groups. CD56 expression was detected in 63/452 (13.9%) patients. CD56(+) T-ALL were predominantly of non-thymic (pre-T 35%, mature 41%) immunophenotypic subtypes, whereas 53% of the CD56(-) cases were thymic T-ALL (p=0.00002). CD13, CD33, CD34 and HLA-DR were significantly more frequently expressed. A clonal T-cell receptor rearrangement was detected in 22/23 CD56(+) ALL. No major clinical differences were observed at presentation. Treatment of CD56(+) ALL resulted in a lower rate of complete remissions (70% vs. 88%) (p=0.001) and a higher rate of resistant disease (21% vs. 8%) (p=0.004). CD56 expression had no significant influence on overall (48% vs. 59%) and disease free survival (67% vs. 57%) at three years. CD56 is expressed on a subset of adult T-ALL with distinct immunophenotypical features and higher resistance to therapy. Most CD56(+) ALL were treated in the high-risk arm of the GMALL study protocols owing to their non-thymic phenotype. Thus after risk adapted treatment a prognostic impact of CD56 expression was not detectable.

Significado pronóstico de los subtipos inmunológicos de la leucemia aguda linfoblástica T del adulto. Estudio de 81 pacientes

T-cell acute lymphoblastic leukemia (ALL) includes 4 immunological subtypes: pro-T, pre-T, thymic or cortical and mature. In some studies, pro-T and mature subtypes have a poor prognosis. The objective of this study was to describe the clinical characteristics, the result of treatment and the prognosis of the immunological subtypes of T-cell ALL in 81 adult patients included in 2 protocols of the Spanish PETHEMA group (ALL-96 and ALL-93). Between 1993 and 2003, 81 adult patients from 22 Spanish hospitals were included in two PETHEMA protocols: ALL-96 for standard-risk patients, and ALL-93 for high- risk patients. The main clinical and biological parameters as well as the rate of response to treatment, the frequency of complete remission , disease free survival and overall survival were compared in each T-cell ALL subtype. Of the 64 evaluable patients the distribution of the immunological subtypes was: 3 pro-T, 17 pre-T, 22 thymic or cortical and 22 mature. Patients with mature T-ALL had higher frequency of central nervous system involvement and myeloid antigen expression than those of the remaining subgroups. Patients with mature T-cell ALL had a slow rate of response to treatment in comparison with patients wit pre-T and mature T-cell ALL but this did not translate to significant differences in frequency of complete remission (77% vs 94%), disease free survival (42% vs 46%) and overall survival (29% vs 47%). Although patients with mature T-cell ALL had a slow rate of response to treatment and their survival tended to be shorter, in the present study there were no statistically significant differences in the prognosis of the different subtypes of T-cell ALL.

Improved Outcome in Adult T-ALL by Risk Adapted Treatment Strategies.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 25% of adult ALL and is characterised by specific clinical and biologic features such as phenotype with early (cyCD3+,CD7+) (E-T), thymic (CD1a+) ) (Thy-T) and mature T-ALL (M-T) (sCD3+). The formerly poor prognosis of T-ALL was more recently improved in some studies albeit not in all, even not in childhood ALL.Patients: To improve outcome of T-ALL the German Multicenter Study Group for Adult ALL initiated two consecutive studies with subtype adapted therapies. 503 T-ALL pts were recruited between 4/93 and 10/03. The median age was 30 (15–55)yrs, 75% were male, 66% had mediastinal tumor (MedTu), 24% WBC > 100.000 and 7% CNS involvement with subtypes as follows: 53% Thy-T, 26% E-T and 21% M-T.Study Design: In Study 05/93 all T-ALL pts were treated uniformly with 8drug induction incl. prophylactic CNS (24 Gy) (CNSRAD) and proph. mediastinal (24 Gy) irradiation (MEDRAD) followed by 7x consolidation (HDAC/MITOX, HDMTX/ASP, reinduction, 2xVM26/AC, 2xCYCLO/AC) and maintenance (6MP/MTX). In Study 06/99 treatment of T-ALL was risk adapted with a shortened, intensified 8drug induction (CNSRAD in all but MEDRAD only in pts with residual MedTu after induction) followed by consolidation I (HDAC/HDMTX/VP16). Thy-T was then treated as standard risk with 6x consolidation (3xHDMTX/ASP,reinduction,VM26/AC,CYCLO/AC). E-T and M-T were considered as high risk and scheduled for stem cell transplantation (SCT) in CR1.Results: In Study 05/93 the CR rate in 291 pts was 89% (94%, 73% and 90% for Thy-T, E-T and M-T; p=.0006) and even 97% for Thy-T in adolescents (15–25 yrs) . Overall 4% failed to achieve CR and 7% died in induction. The probability of continuous CR (CCR) at 5 yrs was overall 53% and 64% for Thy-T, but only 30% (p<.0001) for E-T and M-T. The survival of CR pts (S) was overall 44% and 61%, 19% and 28% (p<.0001) for Thy-T, E-T and M-T. 10% died in CR mainly due to infections - particularly pts with prolonged cytopenias after prior MEDRAD. Immunophenotype was the only prognostic factor confirmed in multivariate analysis. This led to a risk adapted strategy in the subsequent study 06/99. The CR rate in 212 pts was again high (85% overall and 90%, 80% and 76% for subtypes; p=.04). 10% failed to achieve CR and 5% died in induction. 68% of pts with early/mature T-ALL received SCT in CR1 (13 allo sibling, 25 MUD, 11 auto). In these high-risk pts survival after SCT was surprisingly good with 64% at 3 yrs. Survival of all CR pts at 3 yrs was 57% (69% for Thy-T and 50%/34% for E-T/M-T; p=.007). Death in CR did not increase despite SCT (9% overall; 7% after SCT).Conclusion: In a very large cohort of prospectively treated pts excellent results for adult T-ALL could be achieved with subtype adapted strategies. For early/mature T-ALL a doubling of survival of CR pts from 23% to 43% in studis 05/93 versus 06/99 was achieved by SCT. Thus this is the first study showing a substantial benefit of SCT in these subtypes of T-ALL. To improve outcome further, new drugs for T-ALL (e.g. Campath, Cladribine, Nelarabine) are evaluated in relapse pts and in the ongoing GMALL study additional pts for SCT are identified by prospective analysis of minimal residual disease (supported by Deutsche Krebshilfe and partly BMBF, Grant No 01GI 9971)