The genomic basis of childhood T-lineage acute lymphoblastic leukaemia.

T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.

Chronic Hypoxia in an EXTrauterine Environment for Neonatal Development Impairs Lung Development.

Severe fetal hypoxia poses a significant risk to lung development resulting in severe postnatal complications. Existing chronic hypoxia animal models lack the ability to achieve pathologically reduced fetal oxygen without compromising animal development, placental blood flow, or maternal health. Using an established model of isolated chronic hypoxia involving the Extrauterine Environment for Neonatal Development (EXTEND), we are able to investigate the direct impact of fetal hypoxia on lung development. Oxygen delivery to preterm fetal lambs (105-110 days GA) delivered by cesarean section was reduced, and animals were supported on EXTEND through the canalicular or saccular stage of lung development. Fetal lambs in hypoxic conditions showed significant growth restriction compared to their normoxic counterparts. We also observed modest aberrant vascular remodeling in the saccular group after hypoxic conditions with decreased macrovessel numbers, microvascular endothelial cell numbers, and increased peripheral vessel muscularization. In addition, fetal hypoxia resulted in enlarged distal airspaces and decreased septal wall volume. Moreover, there was a reduction in mature SFTPB and processed SFTPC protein expression concomitant with a decrease in AT2 cell number. These findings demonstrate that maternally-independent fetal hypoxia predominantly impacts distal airway development, AT2 cell number, and surfactant production with mild effects on the vasculature.

Inhibition of the Sec61 translocon overcomes cytokine-induced glucocorticoid resistance in T-cell acute lymphoblastic leukaemia.

Glucocorticoid (GC) resistance is a poor prognostic factor in T-cell acute lymphoblastic leukaemia (T-ALL). Interleukin-7 (IL-7) mediates GC resistance via GC-induced upregulation of IL-7 receptor (IL-7R) expression, leading to increased pro-survival signalling. IL-7R reaches the cell surface via the secretory pathway, so we hypothesized that inhibiting the translocation of IL-7R into the secretory pathway would overcome GC resistance. Sec61 is an endoplasmic reticulum (ER) channel that is required for insertion of polypeptides into the ER. Here, we demonstrate that KZR-445, a novel inhibitor of Sec61, potently attenuates the dexamethasone (DEX)-induced increase in cell surface IL-7R and overcomes IL-7-induced DEX resistance.

Rational drug combinations with CDK4/6 inhibitors in acute lymphoblastic leukemia.

Despite improvements in outcomes for children with B- and T-cell acute lymphoblastic leukemia (B-ALL and T-ALL), patients with resistant or relapsed disease fare poorly. Previous studies have demonstrated the essential role of cyclin D3 in T-ALL disease initiation and progression and that targeting of the CDK4/6-cyclin D complex can suppress T-ALL proliferation, leading to efficient cell death in animal models. Studies in leukemia and other malignancies, suggest that schedule is important when combining CDK4/6 inhibitors (CDKi) with cytotoxic agents. Based on these observations, we broadened evaluation of two CDKi, palbociclib (PD-0332991, Pfizer) and ribociclib (LEE011, Novartis) in B- and T-ALL as single agent and in combination with conventional cytotoxic chemotherapy, using different schedules in preclinical models. As monotherapy, CDKi caused cell cycle arrest with a significant decrease in S phase entry and were active in vivo across a broad number of patient-derived xenograft samples. Prolonged monotherapy induces resistance, for which we identified a potential novel mechanism using transcriptome profiling. Importantly, simultaneous but not sequential treatment of CDKi with conventional chemotherapy (dexamethasone, L-asparaginase and vincristine) led to improved efficacy compared to monotherapy in vivo. We provide novel evidence that combining CDKi and conventional chemotherapy can be safe and effective. These results led to the rational design of a clinical trial.

Preclinical efficacy of daratumumab in T-cell acute lymphoblastic leukemia.

As a consequence of acquired or intrinsic disease resistance, the prognosis for patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Novel, less toxic drugs are clearly needed. One of the most promising emerging therapeutic strategies for cancer treatment is targeted immunotherapy. Immune therapies have improved outcomes for patients with other hematologic malignancies including B-cell ALL; however no immune therapy has been successfully developed for T-ALL. We hypothesize targeting CD38 will be effective against T-ALL. We demonstrate that blasts from patients with T-ALL have robust surface CD38 surface expression and that this expression remains stable after exposure to multiagent chemotherapy. CD38 is expressed at very low levels on normal lymphoid and myeloid cells and on a few tissues of nonhematopoietic origin, suggesting that CD38 may be an ideal target. Daratumumab is a human immunoglobulin G1κ monoclonal antibody that binds CD38, and has been demonstrated to be safe and effective in patients with refractory multiple myeloma. We tested daratumumab in a large panel of T-ALL patient-derived xenografts (PDX) and found striking efficacy in 14 of 15 different PDX. These data suggest that daratumumab is a promising novel therapy for pediatric T-ALL patients.

Potent efficacy of combined PI3K/mTOR and JAK or ABL inhibition in murine xenograft models of Ph-like acute lymphoblastic leukemia.

Philadelphia chromosome (Ph)-like B-cell acute lymphoblastic leukemia (Ph-like ALL) is associated with activated JAK/STAT, Abelson kinase (ABL), and/or phosphatidylinositol 3-kinase (PI3K) signaling and poor clinical outcomes. PI3K pathway signaling inhibitors have been minimally investigated in Ph-like ALL. We hypothesized that targeted inhibition of PI3Kα, PI3Kδ, PI3K/mTOR, or target of rapamycin complex 1/2 (TORC1/TORC2) would decrease leukemia proliferation and abrogate aberrant kinase signaling and that combined PI3K pathway and JAK inhibition or PI3K pathway and SRC/ABL inhibition would have superior efficacy compared to inhibitor monotherapy. We treated 10 childhood ALL patient-derived xenograft models harboring various Ph-like genomic alterations with 4 discrete PI3K pathway protein inhibitors and observed marked leukemia reduction and in vivo signaling inhibition in all models. Treatment with dual PI3K/mTOR inhibitor gedatolisib resulted in near eradication of ALL in cytokine receptor-like factor 2 (CRLF2)/JAK-mutant models with mean 92.2% (range, 86.0%-99.4%) reduction vs vehicle controls (P < .0001) and in prolonged animal survival. Gedatolisib also inhibited ALL proliferation in ABL/platelet-derived growth factor receptor (PDGFR)-mutant models with mean 66.9% (range, 42.0%-87.6%) reduction vs vehicle (P < .0001). Combined gedatolisib and ruxolitinib treatment of CRLF2/JAK-mutant models more effectively inhibited ALL proliferation than either inhibitor alone (P < .001) and further enhanced survival. Similarly, superior efficacy of combined gedatolisib and dasatinib was observed in ABL/PDGFR-mutant models (P < .001). Overall, PI3K/mTOR inhibition potently decreased ALL burden in vivo; antileukemia activity was further enhanced with combination inhibitor therapy. Clinical trials testing combinations of kinase inhibitors in Ph-like ALL patients are indicated.

MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia.

Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs 1722 fmol/µg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.

Impact of Genetic Ancestry on T-cell Acute Lymphoblastic Leukemia Outcomes.

The influence of genetic ancestry on biology, survival outcomes, and risk stratification in T-cell Acute Lymphoblastic Leukemia (T-ALL) has not been explored. Genetic ancestry was genomically-derived from DNA-based single nucleotide polymorphisms in children and young adults with T-ALL treated on Children's Oncology Group trial AALL0434. We determined associations of genetic ancestry, leukemia genomics and survival outcomes; co-primary outcomes were genomic subtype, pathway alteration, overall survival (OS), and event-free survival (EFS). Among 1309 patients, T-ALL molecular subtypes varied significantly by genetic ancestry, including increased frequency of genomically defined ETP-like, MLLT10, and BCL11B-activated subtypes in patients of African ancestry. In multivariable Cox models adjusting for high-risk subtype and pathways, patients of Admixed American ancestry had superior 5-year EFS/OS compared with European; EFS/OS for patients of African and European ancestry were similar. The prognostic value of five commonly altered T-ALL genes varied by ancestry - including NOTCH1 , which was associated with superior OS for patients of European and Admixed American ancestry but non-prognostic among patients of African ancestry. Furthermore, a published five-gene risk classifier accurately risk stratified patients of European ancestry, but misclassified patients of African ancestry. We developed a penalized Cox model which successfully risk stratified patients across ancestries. Overall, 80% of patients had a genomic alteration in at least one gene with differential prognostic impact by genetic ancestry. T-ALL genomics and prognostic associations of genomic alterations vary by genetic ancestry. These data demonstrate the importance of incorporating genetic ancestry into analyses of tumor biology for risk classification algorithms.

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia.

PURPOSE: Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. EXPERIMENTAL DESIGN: We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. RESULTS: Typical T-ALL cells had increased dependence on BCL-xL, whereas early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling upregulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. CONCLUSIONS: LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.

CD38 as a pan-hematologic target for chimeric antigen receptor T cells.

Many hematologic malignancies are not curable with chemotherapy and require novel therapeutic approaches. Chimeric antigen receptor (CAR) T-cell therapy is 1 such approach that involves the transfer of T cells engineered to express CARs for a specific cell-surface antigen. CD38 is a validated tumor antigen in multiple myeloma (MM) and T-cell acute lymphoblastic leukemia (T-ALL) and is also overexpressed in acute myeloid leukemia (AML). Here, we developed human CD38-redirected T cells (CART-38) as a unified approach to treat 3 different hematologic malignancies that occur across the pediatric-to-adult age spectrum. Importantly, CD38 expression on activated T cells did not impair CART-38 cells expansion or in vitro function. In xenografted mice, CART-38 mediated the rejection of AML, T-ALL, and MM cell lines and primary samples and prolonged survival. In a xenograft model of normal human hematopoiesis, CART-38 resulted in the expected reduction of hematopoietic progenitors, which warrants caution and careful monitoring of this potential toxicity when translating this new immunotherapy into the clinic. Deploying CART-38 against multiple CD38-expressing malignancies is significant because it expands the potential for this novel therapy to affect diverse patient populations.

Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice.

Invariant NKT (iNKT) cells are innate-like lymphocytes that recognize glycolipid antigens in the context of the MHC class I-like antigen-presenting molecule CD1d. In vivo activation of mouse iNKT cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) results in the acquisition of a hyporesponsive (anergic) phenotype by these cells. Because iNKT cells can become activated in the context of infectious agents, here we evaluated whether iNKT cell activation by microorganisms can influence subsequent responses of these cells to glycolipid antigen stimulation. We found that mouse iNKT cells activated in vivo by multiple bacterial microorganisms, or by bacterial LPS or flagellin, became unresponsive to subsequent activation with alpha-GalCer. This hyporesponsive phenotype of iNKT cells required IL-12 expression and was associated with changes in the surface phenotype of these cells, reduced severity of concanavalin A-induced hepatitis, and alterations in the therapeutic activities of alpha-GalCer. These findings may have important implications for the development of iNKT cell-based therapies.

Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease.

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Glucocorticoids paradoxically facilitate steroid resistance in T cell acute lymphoblastic leukemias and thymocytes.

Glucocorticoids (GCs) are a central component of therapy for patients with T cell acute lymphoblastic leukemia (T-ALL), and although resistance to GCs is a strong negative prognostic indicator in T-ALL, the mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled in the frontline Children's Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cells were cultured in the presence of IL-7, a cytokine that is critical for normal T cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL-7 receptor (IL-7R) expression. In the presence of IL-7, this augmented downstream signal transduction, resulting in increased STAT5 transcriptional output and upregulation of the prosurvival protein BCL-2. Taken together, we showed that IL-7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL-7R/JAK/STAT5/BCL-2 axis.

Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia.

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

Emergence of a Wave of Wnt Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation.

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, in vivo clonal analysis, and ex vivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

Quantitative phosphotyrosine profiling of patient-derived xenografts identifies therapeutic targets in pediatric leukemia.

Activating mutations in tyrosine kinases (TKs) drive pediatric high-risk acute lymphoblastic leukemia (ALL) and confer resistance to standard chemotherapy. Therefore, there is urgent need to characterize dysregulated TK signaling axes in patients with ALL and identify actionable kinase targets for the development of therapeutic strategies. Here, we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. We integrated a quantitative phosphotyrosine profiling method with 'spike-in' stable isotope labeling with amino acids in cell culture (SILAC) and quantified 1394 class I phosphorylation sites in 16 ALL xenografts. Moreover, hierarchical clustering of phosphotyrosine sites could accurately classify these leukemias into either B or T-cell lineages with the high-risk early T-cell precursor (ETP) and Ph-like ALL clustering as a distinct group. Furthermore, we validated this approach by using specific kinase pathway inhibitors to perturb ABL1, FLT3, and JAK TK signaling in four xenografted patient samples. By quantitatively assessing the tyrosine phosphorylation status of activated kinases in xenograft models of ALL, we were able to identify and validate clinically relevant targets. Therefore, this study highlights the application and potential of phosphotyrosine profiling for identifying clinically relevant kinase targets in leukemia.