Patient-derived T-cell Acute Lymphoblastic Leukemia Research Articles

Homoharringtonine inhibits the NOTCH/MYC pathway and exhibits antitumor effects in T-cell acute lymphoblastic leukemia

AbstractWe report on the antileukemic activity of homoharringtonine (HHT) in T-cell acute lymphoblastic leukemia (T-ALL). We showed that HHT inhibited the NOTCH/MYC pathway and induced significantly longer survival in mouse and patient-derived T-ALL xenograft models, supporting HHT as a promising agent for T-ALL.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5920.

Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice

The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

Choline Kinase a Is Required for Alkylating Agent Resistance in T-Cell Acute Lymphoblastic Leukemia

Conventional chemotherapy is responsible for most clinical cures achieved in oncology, but these drugs are toxic, and resistance is a major problem. Indeed,10-20% of children and >50% of adults with T-cell acute lymphoblastic leukemia (T-ALL) exhibit resistance to front-line therapy, leading to a dismal prognosis. Improved treatment options are needed for these patients. To identify mechanisms of alkylating agent resistance, we performed a genome-wide CRISPR screen in T-ALL cells treated with the alkylating agent nitrogen mustard, the active functional group of drugs such as cyclophosphamide and melphalan. The results revealed a number of internal positive controls, including genes implicated in the Fanconi and other DNA repair pathways. This screen also implicated a role for choline kinase A (CHKA) in nitrogen mustard resistance. CHKA is best-known for its ability to catalyze phosphocholine synthesis, a key step in phosphatidylcholine biosynthesis, and has no known role in DNA damage repair. We found that CHKA shRNA knockdown induces profound sensitivity to nitrogen mustard treatment in leukemia cells (p= 0.0012), but not in normal hematopoietic progenitor CD34+ cells. This effect was rescued by restoring expression of wild-type CHKA, but not by an ATPase-deficient CHKA point mutant. This indicates that CHKA kinase activity is required for resistance. We next assessed the role of CHKA in resistance to a panel of therapeutics with distinct mechanisms of action. We found that CHKA loss induced selective sensitivity to drugs that induce bulky DNA adducts. By contrast, CHKA had little effect on drugs that induce other forms of DNA damage, such as free radical damage or double-strand DNA breaks. Studies to elucidate the mechanism through which CHKA mediates resistance to these drugs are underway. To investigate the therapeutic potential of CHKA inhibition, we tested a small molecule CHKA inhibitor (CK2) in combination with alkylating agents in a panel of patient-derived T-ALL models. In vitro, we found that the pharmacologic inhibition of CHKA is synergistic with alkylating agent treatment with a bliss score of 16.683 for PDX9 and 21.982 for PDX CBAT (score >10 indicating strong synergy). We then tested the potential of this approach in these T-ALL PDX models when engrafted in vivo in NRG mice and treated with CK2 and cyclophosphamide alone or in combination. We found that the addition of CK2 further improved the survival benefit of cyclophosphamide in this model (p= 0.0260). Taken together, our findings support a new role of choline kinase A in alkylating agent resistance in T-ALL and provide a rationale to explore the possibility of increasing the efficacy of conventional chemotherapy in the context of drug-resistant leukemias by combining CHKA inhibition with alkylating agents.

P341: A NOVEL BISPECIFIC T CELL ENGAGER (UMG2-CD3) IS EFFECTIVE AGAINST CORTICAL-DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived by T cell precursors and characterized by poor prognosis.The immunotherapy has revolutionized the outcome of B cell acute lymphoblastic leukemia (B-ALL), but the absence of tumor-specific T cell antigen hampers its efficacy in T-ALL. Therefore, the development of novel immune-therapeutic options for the management of this orphan disease is eagerly awaited. CD1a is a glycoprotein expressed on cortical T-ALL and only on healthy thymocytes and Langerhans cells. Taking into account its safe pattern of expression, CD1a might represent a valuable therapeutic target for thetreatment of T-ALL. Aims: With the aim to provide an effective immune-therapeutic strategy for T-ALL, we developed a bispecific T cell engager (BTCE) derived from a novel UMG2 mAb that recognize a previously uncharacterized CD1a epitope. Methods: To evaluate the specificity of UMG2 binding to CD1a epitope, HEK293T cells which do not express CD1a endogenously, have been used. In this regards, cells were transfected with a plasmid that encode for CD1a or with an empty vector and UMG2 reactivity has been evaluated by flow cytometry. To assess the unicity of UMG2 mAb binding, a competitive binding assay between UMG2 and commercially available CD1a antibodies has been performed. The UMG2 expression profile on peripheral blood cells from healthy donors and on a panel of cortical T-ALL cells has been evaluated. To develop a UMG2-CD3 construct, an asymmetric 2 + 1 UMG2-CD3 bispecific T cellengager (BTCE) has been generated by using knobs into hole technology. UMG2-CD3 T cell-redirected cytotoxicity has been evaluated on HEK293T wild type, on HEK293T-CD1a+ and on patient-derived T-ALL cells, co-cultured with peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at different E:T ratio.Moreover, T cell activation has been assesed by flow cytometry. UMG2-CD3 anti-tumor activity against a CD1a+ T-ALL cells has been evaluated in vivo. For this purpose,Hu-PBMCs NSG mouse model has been generated and tumor growth has been assessed by fluorescent imaging probe via IVIS system. Results: UMG2 mAb recognizes a novel CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs with the exception of a partial competition with NA1/34-HLK clone. A strong UMG2 reactivity has been observed on T-ALL cells, while no binding has been found on normal blood cells. A concentration-dependent T cell cytotoxicity on CD1a+ T-ALL cells co-cultured with PBMCs in the presence of UMG2-CD3 has been observed. Minimal UMG2-CD3 residual anti-tumor activity has been observed in CD4/CD8 depleted and CD56 enriched lymphocytes. CD56 depleted and Fc-blocked BMCs were able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-CD3 could not recruit monocytes and NK cells through Fc-FcyR interaction. Moreover, the concentration-dependent increase of i) T cell proliferation, ii) cytotoxic degranulation marker (CD107a), iii) expression of cell surface activation markers (CD25, CD69),and iv) pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ) has been observed in the presence of UMG2-CD3. Importantly, in an in vivo immune-humanized NSG mice model,UMG2-CD3 was able to significantly inhibit tumor growth and then conferring the survival advantage of treated animals. Summary/Conclusion: All our findings, demonstrated that UMG2-CD3 BTCE represents a promising immune-therapeutic agent against T-ALL to be further investigated for clinical translation.

MTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells

mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing.In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia.These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.

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

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

Spleen plays a major role in DLL4-driven acute T-cell lymphoblastic leukemia.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored.Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer.Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model.Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

Potent and Highly Selective Aldo-Keto Reductase 1C3 (AKR1C3) Inhibitors Act as Chemotherapeutic Potentiators in Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the synthesis of 9α,11β-prostaglandin (PG) F2α and PGF2α prostanoids that sustain the growth of myeloid precursors in the bone marrow. The enzyme is overexpressed in acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL). Moreover, AKR1C3 confers chemotherapeutic resistance to the anthracyclines: first-line agents for the treatment of leukemias. The highly homologous isoforms AKR1C1 and AKR1C2 inactivate 5α-dihydrotestosterone, and their inhibition would be undesirable. We report herein the identification of AKR1C3 inhibitors that demonstrate exquisite isoform selectivity for AKR1C3 over the other closely related isoforms to the order of >2800-fold. Biological evaluation of our isoform-selective inhibitors revealed a high degree of synergistic drug action in combination with the clinical leukemia therapeutics daunorubicin and cytarabine in in vitro cellular models of AML and primary patient-derived T-ALL cells. Our developed compounds exhibited >100-fold dose reduction index that results in complete resensitization of a daunorubicin-resistant AML cell line to the chemotherapeutic and >100-fold dose reduction of cytarabine in both AML cell lines and primary T-ALL cells.

The CXCR4 Antagonist, BL8040, Is Highly Active Against Human T-ALL in Preclinical Models

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Approximately 20-25% of pediatric and 50% of adult patients with T-ALL relapse following induction therapy, and the prognosis after relapse is dismal, with 3-year event-free survival of only 10-15%. Thus, there is a clear unmet clinical need for better therapies for T-ALL. CXCL12 (stromal-derived factor-1, SDF1) is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. Here we explore the hypothesis that inhibition of the CXCR4 signaling with a potent new CXCR4 antagonist, BL-8040 alone, or in combination with ABT263 (navitoclax), a BCL2/BCLXL antagonist, will have therapeutic activity against T-ALL.To test this hypothesis, we xenotransplanted a human T-ALL cell line (P12/Ichikawa cells) or 5 different patient-derived T-ALL xenografts into non-irradiated NSG mice. In each case, the T-ALL cells were allowed to engraft and then mice were randomly assigned to one of four treatment groups: 1) vehicle control; 2) BL-8040 alone; 3) ABT263 alone; 4) combined BL-8040 and ABT263. Treatment was given for 2 weeks, and leukemia burden monitored weekly by bioluminescent imaging or by flow cytometry to quantify human CD45+ T-ALL cells in the blood. A minimum of 10 mice in each cohort from two independent experiments were analyzed (Figure 1). A significant reduction in T-ALL burden was observed in all T ALL samples after treatment with BL8040 alone, with a mean fold-decrease compared with control mice after 2 weeks of treatment of 966 ± 651 (range 16-3,486). Consistent with a prior report, the response to ABT263 alone was more variable, with responses seen in 4 of 6 T-ALL samples. All T-ALL samples had a marked response to the combination of BL-8040 and ABT263, with a mean fold decrease of 4,389 ± 2,602 (range, 139-15,199). A previous study suggested that inhibition of CXCR4 signaling may suppress Myc expression (Pitt et al, Cancer Cell, 2015), which is relevant, since overexpression of Myc secondary to mutations in the Notch pathway are common in T-ALL. However, we observed no difference in Myc protein expression or expression of Myc target genes following BL-8040 treatment. Consistent with this observation, treatment with BL8040 had no impact on the cell cycle status of T-ALL cells in vivo. Of note, we observed a marked decrease in Akt and Erk phosphorylation following BL8040, suggesting that CXCR4 signaling may regulate T ALL survival in vivo by suppressing these pathways.Conclusion. Collectively, these data suggest that BL-8040, either alone or in combination with ABT263, is highly active in T-ALL and support our ongoing clinical trial of BL-8040 in combination with nelarabine for patients with relapsed T-ALL (NCT02763384). [Display omitted] DisclosuresUy:GlycoMimetics: Consultancy; Curis: Consultancy. Vainstein:Biolinerx: Employment. Sorani:BioLineRx Ltd.,: Employment. Bohana-Kashtan:BioLineRx Ltd.,: Employment, Equity Ownership; Cell Cure Neurosciences: Equity Ownership. Shaw:BioLineRx Ltd.: Employment, Equity Ownership.

The Nucleotide Kinase Nadk Is Required for ROS Detoxification and Constitutes a Metabolic Vulnerability of NOTCH1-Driven T-ALL

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer with a cumulative risk of ~1 in 2,000 children by the age of 15 years and an increasing incidence over the last 30 years.Whilst advances in the use of combination chemotherapy have significantly improved outcomes, current treatments remain toxic and can have long-term health consequences. T-cell acute lymphoblastic leukemia (T-ALL) represents between 15 to 25% of all ALL and affects both children and adults, and carries a worse prognosis. Activating mutations in the NOTCH1gene are found in more than 60% of cases and are being targeted therapeutically with compounds such as γ-secretase inhibitors (GSIs) and Notch inhibiting antibodies (mAbs). However, none of these approaches has entered mainstream therapy as yet.To address this, we performed a genome wide CRISPR-Cas9 screen in T-ALL cell lines driven by NOTCH1 overexpression (Jurkat, CEM-CCRF), or mutation (PEER) and in a line not dependent on NOTCH1 (Loucy) (Figure 1A). To discover NOTCH1-specific genetic vulnerabilities, we compared essential genes for NOTCH1-dependent T-ALL cell lines with those for Loucy and for 6 human myeloid leukemia cells (MV4;11, MOLM13, OCI-AML2, OCI-AML3, K562 and HL60)1. This identified 69 NOTCH-specific essential genes, including well known players in NOTCH signaling such as NOTCH1, RBPJ, BCL11B, GATA3 and CTPB1(Figure 1B). In addition to these genes, we also identified a separate group of genes associated to cellular pathways involved in the reduction of reactive oxygen species (ROS). One of these was NADK, the gene for nicotinamide adenine dinucleotide kinase, which we investigate further. NADK drives the conversion of NAD+to NADP+, which in its reduced form (NADPH) is used in the reduction of ROS levels through the production of reduced Glutathione. Exposure of Loucy (NOTCH1-WT) to the oxidant H2O2showed that it was not able to reduce intracellular ROS levels as efficiently as NOTCH1-driven lines, suggesting increased NADK activity in the latter. In keeping with this, intracellular NADP+/NADPH levels that were significantly higher in NOTCH1-driven cell lines than in Loucy (Figure 1C). In addition, pharmaceutical or genetic inhibition of NADK led to a significant increase in ROS level in NOTCH1-driven Jurkat cells, while Loucy cells were not affected (Figure 1D). This increase in ROS levels was associated with a significant decrease in cell proliferation (Figure 1E). It was previously reported that, in both human and mouse T-ALL cells, the intracellular domain of Notch1 (NiCD) suppresses production of ROS levels and imparts a higher resistance to H2O2,in so doing facilitating the proliferation of these cells2. We hypothesize that NADK is the mediator of this effect of NOTCH1 and this makes it a therapeutic vulnerability in NOTCH1-driven T-ALL and potentially in other NOTCH1-driven cancers. We currently testing this hypothesis in vivo using patient-derived T-ALL xenografts into immunocompromised mice and investigating the molecular events involved in the NOTCH1-NADK interaction.Our findings propose NADK as a novel therapeutic vulnerability in NOTCH1-driven T-ALL and demonstrate the power genome-wide CRISPR screens in finding novel genetic and therapeutic targets for this disease.1. Tzelepis, K.et al.A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. Cell Rep17, 1193-1205 (2016).2. Giambra, V.et al.NOTCH1 promotes T cell leukemia-initiating activity by RUNX-mediated regulation of PKC-theta and reactive oxygen species. Nat Med18, 1693-8 (2012). [Display omitted] DisclosuresVassiliou:KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding.

Selective killing of human T-ALL cells: an integrated approach targeting redox homeostasis and the OMA1/OPA1 axis

Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients are currently incurable due to primary or secondary resistance to glucocorticoid-based therapies. Here we employed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (ROS) using NS1619, a benzimidazolone that activates the K+ (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients with refractory T-ALL, but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knockdown of the mitochondrial protease OMA1, indicating the engagement of a ROS-OMA1-OPA1 axis in T-ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-induced apoptosis. In vivo, the combination of dexamethasone and NS1619 significantly reduced the growth of a glucocorticoid-resistant patient-derived T-ALL xenograft. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL.

Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies.

Effective immunotherapies for T-cell malignancies are lacking. We devised a novel approach based on chimeric antigen receptor (CAR)-redirected T lymphocytes. We selected CD7 as a target because of its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL samples), median CD7 expression was >99%; CD7 expression remained high at relapse (n = 14), and during chemotherapy (n = 54). We targeted CD7 with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes caused fratricide due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control fratricide, we applied a new method (protein expression blocker [PEBL]), based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain. Transduction of anti-CD7 PEBL resulted in virtually instantaneous abrogation of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7+ vs 98.1% ± 1.5% of mock-transduced T cells (n = 5; P < .0001). PEBL expression did not impair T-cell proliferation, interferon-γ and tumor necrosis factor-α secretion, or cytotoxicity, and eliminated CAR-mediated fratricide. PEBL-CAR T cells were highly cytotoxic against CD7+ leukemic cells in vitro and were consistently more potent than CD7+ T cells spared by fratricide. They also showed strong anti-leukemic activity in cell line- and patient-derived T-ALL xenografts. The strategy described in this study fits well with existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.

Efficacy of the CDK inhibitor dinaciclib in vitro and in vivo in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease of the blood affecting children, adolescents and adults. Although current treatment protocols for T-ALL have improved overall survival, a portion of T-ALL patients still experiences treatment failure. Thus, the development of novel therapies is needed. In this study, we used several patient-derived T-ALL cell lines to screen for an effective drug for T-ALL. Using a panel of 378 inhibitors against different kinases, we identified the CDK inhibitor dinaciclib as a potential drug for T-ALL. Dinaciclib treatment significantly reduced cell viability and completely blocked colony formation. Furthermore, cells treated with dinaciclib showed decreased expression of several pro-survival proteins including survivin, cyclin T1 and c-MYC. Dinaciclib treatment also increased accumulation of cells in G2/M phase and significantly induced apoptosis. Finally, dinaciclib extended survival of mice in a T-ALL cell xenograft model. Collectively, these data suggest that the CDK inhibitor dinaciclib is an active drug for T-ALL in the preclinical settings.

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential.

BackgroundNanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II.Methods and resultsThree new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing.ConclusionWe have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia.

Anti-Leukemic Activity of the TYK2 Selective Inhibitor Ndi-031301 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the transformation of T-cell progenitors. Although the prognosis of this disease has substantially improved due to the introduction of intensified chemotherapy, the clinical outcome of T-ALL patients with primary resistant or relapsed disease remains poor, indicating that further therapeutic improvement is urgently needed. We have previously demonstrated that activation of tyrosine kinase 2 (TYK2) contributes to aberrant survival of human T-ALL cells. TYK2 is a member of the Janus-activated kinase (JAK) tyrosine kinase family and our report was the first to implicate TYK2 in T-ALL pathogenesis. Indeed, our gene knockdown experiments showed TYK2 dependency in 14 of 16 (88%) T-ALL cell lines and 5 of 8 (63%) patient-derived T-ALL cells tested, suggesting that inhibition of TYK2 would be beneficial in most patients with T-ALL. Based on these findings, we investigated the therapeutic potential of a novel small-molecule TYK2 kinase inhibitor NDI-031301 in T-ALL. We found that NDI-031301 shows potent and selective inhibitory activity against TYK2 in a cellular context, because this compound strongly inhibited the growth of TYK2-transfomed Ba/F3 cells when compared to the JAK inhibitors tofacitinib and baricitinib, whereas Ba/F3 cells transformed by other tyrosine kinases showed decreased sensitivity to NDI-031301. NDI-031301 induced robust growth inhibition in each of 4 human T-ALL cell lines representing different molecular subtypes of the disease (DU.528, KOPT-K1, HPB-ALL and SKW-3), with IC50 values of 0.8186 - 2.380 μM after 72 hours of exposure. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with tofacitinib and baricitinib. To elucidate the mechanism of apoptosis induced by NDI-031301 in T-ALL cells, we next investigated cellular signaling pathways that are associated with cell survival and specifically affected by TYK2 inhibition with NDI-031301. Western blotting analysis demonstrated that treatment with 3 μM of NDI-031301 resulted in reduction of STAT1 Tyr-701 phosphorylation and BCL2 levels in KOPT-K1 cells, consistent with our previous finding that TYK2 phosphorylates STAT1 and upregulates BCL2 expression in most T-ALL cells. Surprisingly, the treatment also uniquely led to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage, which was not observed with tofacitinib and baricitinib. NDI-031301-mediated activation of SAPK/JNK and p38 MAPK pathways are likely mediated through inhibition of TYK2, because increased phosphorylation levels of SAPK/JNK and p38 MAPK were observed in the cells transfected with TYK2-targeting shRNAs, while the levels of ERK1/2 phosphorylation were not upregulated. Further investigation revealed that activation of p38 MAPK occurred within 1 hour of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacologic inhibition of p38 MAPK by SB203580 partially rescued apoptosis induced by TYK2 inhibitor, while inhibition of ERK or SAPK/JNK showed no rescue effects. Finally, we found that daily oral administration of NDI-031301 at 100mg/kg BID to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumor burden and a significant survival benefit. After 29 days of treatment, the mice receiving NDI-031301 had marked reductions in infiltration of leukemia cells into spleen and bone marrow by comparison with controls. Thus, our findings clearly support TYK2 inhibition with NDI-031301 or a related compound as a potential therapeutic strategy for patients with T-ALL, and also raise the possibility that enhancing p38 MAPK activation in T-ALL cells may be an approach to accentuate its anti-leukemic activity. DisclosuresMasse:Nimbus Therapeutics: Employment. Miao:Nimbus Therapeutics: Employment. Rocnik:Nimbus Therapeutics: Employment. Kapeller:Nimbus Therapeutics: Employment.

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

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. 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.

Abstract LB-210: KPT-8602 is a second-generation XPO1 inhibitor with improved in vivo tolerability that demonstrates potent acute lymphoblastic leukemia activity

Abstract Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a wide variety of different cargo proteins including tumor suppressors out of the cell's nucleus. Inhibition of XPO1 function consequently restores nuclear localization of these proteins and is a promising therapeutic strategy for cancer. Selective inhibitor of nuclear export (SINE) compounds are inhibitors of the XPO1-mediated nuclear export with potent anti-cancer activity. The oral clinical candidate SINE selinexor (KPT-330) is currently in Phase-II/IIb clinical trials and demonstrated remission in patients as a single agent or in combination therapy in trials for pre-treated, relapsed and refractory hematological and solid tumor malignancies. The drug is generally well tolerated when dosed every other day 1-3 times a week. However, XPO1 inhibitors with improved tolerability allowing more frequent dosing can be expected to have a substantial clinical benefit. Here we present the anti-leukemic activity of a second-generation XPO1 inhibitor KPT-8602 with improved tolerability allowing for a daily dosing regimen. First, its anti-XPO1 activity was assessed; KPT-8602 potently inhibited the XPO1-mediated nuclear protein export at nanomolar concentrations and it blocked the interaction of XPO1 with cargo protein. KPT-8602 also induced potent cytotoxicity on a panel of T-ALL and B-ALL cell lines. Cytotoxicity correlated with the induction of caspase-dependent apoptosis and the nuclear accumulation of p53 as well as the subsequent induction of p53 response. To further investigate the mechanism of action of KPT-8602 we applied CRISPR/Cas9 to introduce a Cys528Ser mutation in the XPO1 gene of four different leukemia cell lines. Mutant cells were over 100 times resistant to KPT-8602. In addition, drug-target interaction was confirmed by pull-down of wild-type XPO1 protein out of cells using biotinylated KPT-8602 while it was unable to pull down mutated XPO1C528S out of mutant cells. These results illustrate the highly specific interaction of the drug for its target and prove that the anti-leukemic activity of KPT-8602 is caused by inhibition of XPO1. To examine the anti-leukemic activity of KPT-8062 in vivo, mice engrafted with patient-derived T-cell acute lymphoblastic leukemia were treated with KPT-8602 or placebo. Mice were daily treated by oral gavage for 3 weeks. Treatment with KPT-8602 led to a significant reduction of leukemia cell numbers in blood as measured by weekly blood counts, without affecting normal erythropoiesis. Animals treated with KPT-8602 had prolonged survival compared to placebo treated animals. KPT-8602 also showed potent anti-leukemia activity in a mouse T-ALL leukemia model. In conclusion, KPT-8062 is a second-generation XPO1 inhibitor with high specificity for its target and with potent anti-ALL activity. It displays better tolerability as compared to the first-generation SINE selinexor allowing it for daily dosing resulting in effective anti-ALL activity in in vivo PDX models warranting further evaluation of this new drug in patients. As such, in January 2016 a phase I/II study with KPT-8602 has been initiated. Citation Format: Dirk Daelemans, Jasper Edgar Neggers, Jolien De Bie, Maarten Jacquemyn, Astrid D’Hoore, Els Vanstreels, Erkan Baloglu, Yosef Landesman, Sharon Shacham, William Senapedis, Antonis Dagklis, Thomas Vercruysse, Jan Cools. KPT-8602 is a second-generation XPO1 inhibitor with improved in vivo tolerability that demonstrates potent acute lymphoblastic leukemia activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-210.

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia.

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient's cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.

A pre-clinical model of resistance to induction therapy in pediatric acute lymphoblastic leukemia.

Relapse and acquired drug resistance in T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. This study was designed to establish a preclinical model of resistance to induction therapy in childhood T-ALL to examine the emergence of drug resistance and identify novel therapies. Patient-derived T-ALL xenografts in immune-deficient (non-obese diabetic/severe combined immunodeficient) mice were exposed to a four-drug combination of vincristine, dexamethasone (DEX), L-asparaginase and daunorubicin (VXLD). ‘Relapse' xenografts were characterized by responses to drugs, changes in gene expression profiles and Connectivity Map (CMap) prediction of strategies to reverse drug resistance. Two of four xenografts developed ex vivo and in vivo drug resistance. Both resistant lines showed altered lipid and cholesterol metabolism, yet they had a distinct drug resistance pattern. CMap analyses reinforced these features, identifying the cholesterol pathway inhibitor simvastatin (SVT) as a potential therapy to overcome resistance. Combined ex vivo with DEX, SVT was significantly synergistic, yet when administered in vivo with VXLD it did not delay leukemia progression. Synergy of SVT with established chemotherapy may depend on higher drug doses than are tolerable in this model. Taken together, we have developed a clinically relevant in vivo model of T-ALL suitable to examine the emergence of drug resistance and to identify novel therapies.