Targeting the TNF/IAP pathway synergizes with anti-CD3 immunotherapy in T-cell acute lymphoblastic leukemia.

ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Current treatments, based on intensive chemotherapy regimens provide overall survival rates of ∼85% in children and <50% in adults, calling the search of new therapeutic options. We previously reported that targeting the T-cell receptor (TCR) in T-ALL with anti-CD3 (αCD3) monoclonal antibodies (mAbs) enforces a molecular program akin to thymic negative selection, a major developmental checkpoint in normal T-cell development; induces leukemic cell death; and impairs leukemia progression to ultimately improve host survival. However, αCD3 monotherapy resulted in relapse. To find out actionable targets able to re-enforce leukemic cells' vulnerability to αCD3 mAbs, including the clinically relevant teplizumab, we identified the molecular program induced by αCD3 mAbs in patient-derived xenografts derived from T-ALL cases. Using large-scale transcriptomic analysis, we found prominent expression of tumor necrosis factor α (TNFα), lymphotoxin α (LTα), and multiple components of the "TNFα via NF-κB signaling" pathway in anti-CD3-treated T-ALL. We show in vivo that etanercept, a sink for TNFα/LTα, enhances αCD3 antileukemic properties, indicating that TNF/TNF receptor (TNFR) survival pathways interferes with TCR-induced leukemic cell death. However, suppression of TNF-mediated survival and switch to TNFR-mediated cell death through inhibition of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) with the second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant synergizes with αCD3 to impair leukemia expansion in a receptor-interacting serine/threonine-protein kinase 1-dependent manner and improve mice survival. Thus, our results advocate the use of either TNFα/LTα inhibitors, or birinapant/other SMAC mimetics to improve anti-CD3 immunotherapy in T-ALL.

Harnessing the MYB-dependent TAL1 5'super-enhancer for targeted therapy in T-ALL.

The acquisition of genetic abnormalities engendering oncogene dysregulation underpins cancer development. Certain proto-oncogenes possess several dysregulation mechanisms, yet how each mechanism impacts clinical outcome is unclear. Using T-cell acute lymphoblastic leukemia (T-ALL) as an example, we show that patients harboring 5'super-enhancer (5'SE) mutations of the TAL1 oncogene identifies a specific patient subgroup with poor prognosis irrespective of the level of oncogene dysregulation. Remarkably, the MYB dependent oncogenic 5'SE can be targeted using Mebendazole to induce MYB protein degradation and T-ALL cell death. Of note Mebendazole treatment demonstrated efficacy in vivo in T-ALL preclinical models. Our work provides proof of concept that within a specific oncogene driven cancer, the mechanism of oncogene dysregulation rather than the oncogene itself can identify clinically distinct patient subgroups and pave the way for future super-enhancer targeting therapy.

Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL).

Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.

NFAT transcription factors are essential and redundant actors for leukemia initiating potential in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few available targeted therapies. We previously reported that the phosphatase calcineurin (Cn) is required for LIC (leukemia Initiating Capacity) potential of T-ALL pointing to Cn as an interesting therapeutic target. Calcineurin inhibitors have however unwanted side effect. NFAT transcription factors play crucial roles downstream of calcineurin during thymocyte development, T cell differentiation, activation and anergy. Here we elucidate NFAT functional relevance in T-ALL. Using murine T-ALL models in which Nfat genes can be inactivated either singly or in combination, we show that NFATs are required for T-ALL LIC potential and essential to survival, proliferation and migration of T-ALL cells. We also demonstrate that Nfat genes are functionally redundant in T-ALL and identified a node of genes commonly deregulated upon Cn or NFAT inactivation, which may serve as future candidate targets for T-ALL.

AXL Inhibition in Macrophages Stimulates Host-versus-Leukemia Immunity and Eradicates Naïve and Treatment-Resistant Leukemia.

Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell- and T cell-dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1-refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells. SIGNIFICANCE: Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy.This article is highlighted in the In This Issue feature, p. 2659.

Epigenetic analysis of patients with T-ALL identifies poor outcomes and a hypomethylating agent-responsive subgroup.

Adult "T cell" acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is associated with poor outcomes, requiring additional therapeutic options. The DNA methylation landscapes of adult T-ALL remain undercharacterized. Here, we systematically analyzed the DNA methylation profiles of normal thymic-sorted T cell subpopulations and 143 primary adult T-ALLs as part of the French GRAALL 2003-2005 trial. Our results indicated that T-ALL is epigenetically heterogeneous consisting of five subtypes (C1-C5), which were either associated with co-occurring DNA methyltransferase 3 alpha (DNMT3A)/isocitrate dehydrogenase [NADP(+)] 2 (IDH2) mutations (C1), TAL bHLH transcription factor 1, erythroid differentiation factor (TAL1) deregulation (C2), T cell leukemia homeobox 3 (TLX3) (C3), TLX1/in cis-homeobox A9 (HOXA9) (C4), or in trans-HOXA9 overexpression (C5). Integrative analysis of DNA methylation and gene expression identified potential cluster-specific oncogenes and tumor suppressor genes. In addition to an aggressive hypomethylated subgroup (C1), our data identified an unexpected subset of hypermethylated T-ALL (C5) associated with poor outcome and primary therapeutic response. Using mouse xenografts, we demonstrated that hypermethylated T-ALL samples exhibited therapeutic responses to the DNA hypomethylating agent 5-azacytidine, which significantly (survival probability; P = 0.001 for C3, 0.01 for C4, and 0.0253 for C5) delayed tumor progression. These findings suggest that epigenetic-based therapies may provide an alternative treatment option in hypermethylated T-ALL.

Preclinical efficacy of humanized, non-FcγR-binding anti-CD3 antibodies in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for ∼20% of ALL cases. Intensive chemotherapy regimens result in cure rates >85% in children and <50% in adults, warranting a search of novel therapeutic strategies. Although immune-based therapies have tremendously improved the treatment of B-ALL and other B-cell malignancies, they are not yet available for T-ALL. We report here that humanized, non-Fcγ receptor (FcγR)-binding monoclonal antibodies (mAbs) to CD3 have antileukemic properties in xenograft (PDX) models of CD3+ T-ALL, resulting in prolonged host survival. We also report that these antibodies cooperate with chemotherapy to enhance antileukemic effects and host survival. Because these antibodies show only minor, manageable adverse effects in humans, they offer a new therapeutic option for the treatment of T-ALL. Our results also show that the antileukemic properties of anti-CD3 mAbs are largely independent of FcγR-mediated pathways in T-ALL PDXs.

The TCR/CD3 complex in leukemogenesis and as a therapeutic target in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) arises from T cell precursors and is characterized by expression of many lineage-specific proteins. While T-cell antigen receptor (TCR) signaling and its strength are central for thymocyte development, mature T cell homeostasis and immune responses, their roles in T-ALL remain undetermined. Indeed, in contrast to mouse models, in which absence of TCR or major histocompatibility complex binding does not impact on leukemogenesis, other mouse models suggest that basal or weak signaling drives leukemia development. However, recent reports indicate that strong TCR signaling can be detrimental to leukemic cells. Indeed, sustained/high level TCR signaling, stimulated by antigen or CD3 antibody, is strongly anti-leukemic in both murine T-ALL expressing endogenous or transgenic TCR and diagnostic T-ALL cases. As discussed, further work should address the efficacy of T-ALL therapeutic targeting with either TCR/CD3 antibodies or TCR-directed chimeric antigen receptor T cells.

Novel Intergenically Spliced Chimera, NFATC3-PLA2G15, Is Associated with Aggressive T-ALL Biology and Outcome.

Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens (n = 24), and candidate T-ALL-related ISCs were identified (n = 55; a median of 4/patient). In-depth characterization of the NFATC3-PLA2G15 chimera, which was variably expressed in primary T-ALL, was performed. Functional assessment revealed that the fusion had lower activity than wild-type NFATC3 in vitro, and T-ALLs with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT pathway genes in vivo Strikingly, high expression of the NFATC3-PLA2G15 chimera correlated with aggressive disease biology in murine patient-derived T-ALL xenografts, and poor prognosis in human T-ALL patients. Mol Cancer Res; 16(3); 470-5. ©2018 AACR.

Triggering the TCR Developmental Checkpoint Activates a Therapeutically Targetable Tumor Suppressive Pathway in T-cell Leukemia.

UNLABELLED: Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. SIGNIFICANCE: T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932.

Microenvironmental cues for T-cell acute lymphoblastic leukemia development.

Intensive chemotherapy regimens have led to a substantial improvement in the cure rate of patients suffering from T-cell acute lymphoblastic leukemia (T-ALL). Despite this progress, about 15% and 50% of pediatric and adult cases, respectively, show resistance to treatment or relapse with dismal prognosis, calling for further therapeutic investigations. T-ALL is an heterogeneous disease, which presents intrinsic alterations leading to aberrant expression of transcription factors normally involved in hematopoietic stem/progenitor cell development and mutations in genes implicated in the regulation of cell cycle progression, apoptosis, and T-cell development. Gene expression profiling allowed the classification of T-ALL into defined molecular subgroups that mostly reflects the stage of their differentiation arrest. So far this knowledge has not translated into novel, targeted therapy. Recent evidence points to the importance of extrinsic signaling cues in controlling the ability of T-ALL to home, survive, and proliferate, thus offering the perspective of new therapeutic options. This review summarizes the present understanding of the interactions between hematopoietic cells and bone marrow/thymic niches during normal hematopoiesis, describes the main signaling pathways implicated in this dialog, and finally highlights how malignant T cells rely on specific niches to maintain their ability to sustain and propagate leukemia.

Lymphotoxin-ß receptor in microenvironmental cells promotes the development of T-cell acute lymphoblastic leukaemia with cortical/mature immunophenotype.

Lymphotoxin-mediated activation of the lymphotoxin-ß receptor (LTßR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTß (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 ß2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTßR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTßR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTßR signalling in thymic stromal cells, thus promoting leukaemogenesis.

CXCR4 Is Required for Leukemia-Initiating Cell Activity in T Cell Acute Lymphoblastic Leukemia.

Impaired cell migration has been demonstrated in T cell acute lymphoblastic leukemia (T-ALL) cells upon calcineurin inactivation, among other phenotypic traits including increased apoptosis, inhibition of cell proliferation, and ultimately inhibition of leukemia-initiating cell (LIC) activity. Herein we demonstrate that the chemokine receptor CXCR4 is essential to the LIC activity of T-ALL leukemic cells both in NOTCH-induced mouse T-ALL and human T-ALL xenograft models. We further demonstrate that calcineurin regulates CXCR4 cell-surface expression in a cortactin-dependent manner, a mechanism essential to the migratory properties of T-ALL cells. Because 20%-25% of pediatric and over 50% of adult patients with T-ALL do not achieve complete remission and relapse, our results call for clinical trials incorporating CXCR4 antagonists in T-ALL treatment.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

Interleukin-18 produced by bone marrow-derived stromal cells supports T-cell acute leukaemia progression.

Development of novel therapies is critical for T-cell acute leukaemia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleukin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xenografted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.

Lethal giant larvae 1 tumour suppressor activity is not conserved in models of mammalian T and B cell leukaemia.

In epithelial and stem cells, lethal giant larvae (Lgl) is a potent tumour suppressor, a regulator of Notch signalling, and a mediator of cell fate via asymmetric cell division. Recent evidence suggests that the function of Lgl is conserved in mammalian haematopoietic stem cells and implies a contribution to haematological malignancies. To date, direct measurement of the effect of Lgl expression on malignancies of the haematopoietic lineage has not been tested. In Lgl1⁻/⁻ mice, we analysed the development of haematopoietic malignancies either alone, or in the presence of common oncogenic lesions. We show that in the absence of Lgl1, production of mature white blood cell lineages and long-term survival of mice are not affected. Additionally, loss of Lgl1 does not alter leukaemia driven by constitutive Notch, c-Myc or Jak2 signalling. These results suggest that the role of Lgl1 in the haematopoietic lineage might be restricted to specific co-operating mutations and a limited number of cellular contexts.

Combined targeting of JAK2 and Bcl-2/Bcl-xL to cure mutant JAK2-driven malignancies and overcome acquired resistance to JAK2 inhibitors.

To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2) and the effector stage (Bcl-2/Bcl-xL), is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.