Noncoding mutations drive persistence of a founder preleukemic clone which initiates late relapse in T-ALL.

ABSTRACT: T-ALL relapse usually occurs early but can occur much later, which has been suggested to represent a de novo leukemia. However, we conclusively demonstrate late relapse can evolve from a pre-leukemic subclone harbouring a non-coding mutation that evades initial chemotherapy.

Anti-CCR9 chimeric antigen receptor T cells for T-cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes, associated with higher rates of induction failure compared with those in B cell acute lymphoblastic leukemia. The potent immunotherapeutic approaches applied in B cell acute lymphoblastic leukemia, which have revolutionized the treatment paradigm, have proven more challenging in T-ALL, largely due to a lack of target antigens expressed on malignant but not healthy T cells. Unlike B cell depletion, T-cell aplasia is highly toxic. Here, we show that the chemokine receptor CCR9 is expressed in >70% of cases of T-ALL, including >85% of relapsed/refractory disease, and only on a small fraction (<5%) of normal T cells. Using cell line models and patient-derived xenografts, we found that chimeric antigen receptor (CAR) T-cells targeting CCR9 are resistant to fratricide and have potent antileukemic activity both in vitro and in vivo, even at low target antigen density. We propose that anti-CCR9 CAR-T cells could be a highly effective treatment strategy for T-ALL, avoiding T cell aplasia and the need for genome engineering that complicate other approaches.

Targeting hyperactive platelet-derived growth factor receptor-ß signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T cell acute lymphoblastic leukemia (T-ALL) and T cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematological malignancies. Current treatment consists of intensive chemotherapy, leading to 80% overall survival but are associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL and that screening T-ALL/TLBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Targeting cytokine- and therapy-induced PIM1 activation in preclinical models of T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

Current and emerging therapeutic approaches for T-cell acute lymphoblastic leukaemia.

T-cell ALL (T-ALL) is an aggressive malignancy of T-cell progenitors. Although survival outcomes in T-ALL have greatly improved over the past 50 years, relapsed and refractory cases remain extremely challenging to treat and those who cannot tolerate intensive treatment continue to have poor outcomes. Furthermore, T-ALL has proven a more challenging immunotherapeutic target than B-ALL. In this review we explore our expanding knowledge of the basic biology of T-ALL and how this is paving the way for repurposing established treatments and the development of novel therapeutic approaches.

Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress.

We investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)-inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the "rescue" function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell-derived ALL, wherein CD19+ cells closely associated with nestin+ MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Notch signaling: switching an oncogene to a tumor suppressor.

The Notch signaling pathway is a regulator of self-renewal and differentiation in several tissues and cell types. Notch is a binary cell-fate determinant, and its hyperactivation has been implicated as oncogenic in several cancers including breast cancer and T-cell acute lymphoblastic leukemia (T-ALL). Recently, several studies also unraveled tumor-suppressor roles for Notch signaling in different tissues, including tissues where it was before recognized as an oncogene in specific lineages. Whereas involvement of Notch as an oncogene in several lymphoid malignancies (T-ALL, B-chronic lymphocytic leukemia, splenic marginal zone lymphoma) is well characterized, there is growing evidence involving Notch signaling as a tumor suppressor in myeloid malignancies. It therefore appears that Notch signaling pathway's oncogenic or tumor-suppressor abilities are highly context dependent. In this review, we summarize and discuss latest advances in the understanding of this dual role in hematopoiesis and the possible consequences for the treatment of hematologic malignancies.

Novel activating mutations lacking cysteine in type I cytokine receptors in acute lymphoblastic leukemia.

Gain-of-function somatic mutations introducing cysteines to either the extracellular or to the transmembrane domain (TMD) in interleukin-7 receptor α (IL7R) or cytokine receptor-like factor 2 (CRLF2) have been described in acute lymphoblastic leukemias. Here we report noncysteine in-frame mutations in IL7R and CRLF2 located in a region of the TMD closer to the cytosolic domain. Biochemical and functional assays showed that these are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells in vitro and are transforming in vivo. Protein fragment complementation assays suggest that despite the absence of cysteines, the mechanism of activation is through ligand-independent dimerization. Mutagenesis experiments and ConSurf calculations suggest that the mutations stabilize the homodimeric conformation, positioning the cytosolic kinases in predefined orientation to each other, thereby inducing spontaneous receptor activation independently of external signals. Hence, type I cytokine receptors may be activated in leukemia through 2 types of transmembrane somatic dimerizing mutations.

Targeting oncogenic interleukin-7 receptor signalling with N-acetylcysteine in T cell acute lymphoblastic leukaemia.

Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T-ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R-mutant DND-41 cells as assessed by non-reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND-41 cells, and Ba/F3 cells transformed by an IL7R-mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND-41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T-ALL.

Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in pancreatic ductal adenocarcinoma cells.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) involves activation of c-Ki-ras2 Kirsten rat sarcoma oncogene homolog (KRAS) signaling, but little is known about the roles of proteins that regulate the activity of oncogenic KRAS. We investigated the activities of proteins that interact with KRAS in PDAC cells. METHODS: We used mass spectrometry to demonstrate that heterogeneous nuclear ribonucleoproteins (HNRNP) A2 and B1 (encoded by the gene HNRNPA2B1) interact with KRAS G12V. We used co-immunoprecipitation analyses to study interactions between HNRNPA2B1 and KRAS in KRAS-dependent and KRAS-independent PDAC cell lines. We knocked down HNRNPA2B1 using small hairpin RNAs and measured viability, anchorage-independent proliferation, and growth of xenograft tumors in mice. We studied KRAS phosphorylation using the Phos-tag system. RESULTS: We found that interactions between HRNPA2B1 and KRAS correlated with KRAS-dependency of some human PDAC cell lines. Knock down of HNRNPA2B1 significantly reduced viability, anchorage-independent proliferation, and formation of xenograft tumors by KRAS-dependent PDAC cells. HNRNPA2B1 knock down also increased apoptosis of KRAS-dependent PDAC cells, inactivated c-akt murine thymoma oncogene homolog 1 signaling via mammalian target of rapamycin, and reduced interaction between KRAS and phosphatidylinositide 3-kinase. Interaction between HNRNPA2B1 and KRAS required KRAS phosphorylation at serine 181. CONCLUSIONS: In KRAS-dependent PDAC cell lines, HNRNPA2B1 interacts with and regulates the activity of KRAS G12V and G12D. HNRNPA2B1 is required for KRAS activation of c-akt murine thymoma oncogene homolog 1-mammalian target of rapamycin signaling, interaction with phosphatidylinositide 3-kinase, and PDAC cell survival and tumor formation in mice. HNRNPA2B1 might be a target for treatment of pancreatic cancer.

KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia.

This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising 'first-in-class' drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML.

The Clinicogenomic Landscape of Induction Failure in Childhood and Young Adult T-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Failure to respond to induction chemotherapy portends a poor outcome in childhood acute lymphoblastic leukemia (ALL) and is more frequent in T-cell ALL (T-ALL) than B-cell ALL. We aimed to address the limited understanding of clinical and genetic factors that influence outcome in a cohort of patients with T-ALL induction failure (IF). METHODS: We studied all cases of T-ALL IF on two consecutive multinational randomized trials, UKALL2003 and UKALL2011, to define risk factors, treatment, and outcomes. We performed multiomic profiling to characterize the genomic landscape. RESULTS: IF occurred in 10.3% of cases and was significantly associated with increasing age, occurring in 20% of patients age 16 years and older. Five-year overall survival (OS) rates were 52.1% in IF and 90.2% in responsive patients (P < .001). Despite increased use of nelarabine-based chemotherapy consolidated by hematopoietic stem-cell transplant in UKALL2011, there was no improvement in outcome. Persistent end-of-consolidation molecular residual disease resulted in a significantly worse outcome (5-year OS, 14.3% v 68.5%; HR, 4.10; 95% CI, 1.35 to 12.45; P = .0071). Genomic profiling revealed a heterogeneous picture with 25 different initiating lesions converging on 10 subtype-defining genes. There was a remarkable abundance of TAL1 noncoding lesions, associated with a dismal outcome (5-year OS, 12.5%). Combining TAL1 lesions with mutations in the MYC and RAS pathways produces a genetic stratifier that identifies patients highly likely to fail conventional therapy (5-year OS, 23.1% v 86.4%; HR, 6.84; 95% CI, 2.78 to 16.78; P < .0001) and who should therefore be considered for experimental agents. CONCLUSION: The outcome of IF in T-ALL remains poor with current therapy. The lack of a unifying genetic driver suggests alternative approaches, particularly using immunotherapy, are urgently needed.

Pre-Clinical Evaluation of the Hypomethylating Agent Decitabine for the Treatment of T-Cell Lymphoblastic Lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

Therapeutic targeting of "undruggable" MYC.

c-MYC controls global gene expression and regulates cell proliferation, cell differentiation, cell cycle, metabolism and apoptosis. According to some estimates, MYC is dysregulated in ≈70% of human cancers and strong evidence implicates aberrantly expressed MYC in both tumor initiation and maintenance. In vivo studies show that MYC inhibition elicits a prominent anti-proliferative effect and sustained tumor regression while any alteration on healthy tissue remains reversible. This opens an exploitable window for treatment that makes MYC one of the most appealing therapeutic targets for cancer drug development. This review describes the main functional and structural features of the protein structure of MYC and provides a general overview of the most relevant or recently identified interactors that modulate MYC oncogenic activity. This review also summarizes the different approaches aiming to abrogate MYC oncogenic function, with a particular focus on the prototype inhibitors designed for the direct and indirect targeting of MYC.

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.