T-ALL Mice Research Articles

An Anti-CD7 Antibody-Drug Conjugate Target Showing Potent Antitumor Activity for T-Lymphoblastic Leukemia (T-ALL).

Acute T-lymphoblastic leukemia (T-ALL) is a type of leukemia that can occur in both pediatric and adult populations. Compared to acute B-cell lymphoblastic leukemia (B-ALL), patients with T-cell T-ALL have a poorer therapeutic efficacy. In this study, a novel anti-CD7 antibody-drug conjugate (ADC, J87-Dxd) was successfully generated and used for T-ALL treatment. Firstly, to obtain anti-CD7 mAbs, we expressed and purified the CD7 protein extracellular domain. Utilizing hybridoma technology, we obtained three anti-CD7 mAbs (J87, G73 and A15) with a high affinity for CD7. Both the results of immunofluorescence and Biacore assay indicated that J87 (KD = 1.54 × 10-10 M) had the highest affinity among the three anti-CD7 mAbs. In addition, an internalization assay showed the internalization level of J87 to be higher than that of the other two mAbs. Next, we successfully generated the anti-CD7 ADC (J87-Dxd) by conjugating DXd to J87 via a cleavable maleimide-GGFG peptide linker. J87-Dxd also possessed the ability to recognize and bind CD7. Using J87-Dxd to treat T-ALL cells (Jurkat and CCRF-CEM), we observed that J87-Dxd bound to CD7 was internalized into T-ALL cells. Moreover, J87-Dxd treatment significantly induced the apoptosis of Jurkat and CCRF-CEM cells. The IC50 (half-maximal inhibitory concentration) value of J87-Dxd against CCRF-CEM obtained by CCK-8 assay was 6.3 nM. Finally, to assess the antitumor efficacy of a J87-Dxd in vivo, we established T-ALL mouse models and treated mice with J87-Dxd or J87. The results showed that on day 24 after tumor inoculation, all mice treated with J87 or PBS died, whereas the survival rate of mice treated with J87-Dxd was 80%. H&E staining showed no significant organic changes in the heart, liver, spleen, lungs and kidneys of all mice. In summary, we demonstrated that the novel anti-CD7 ADC (J87-Dxd) had a potent and selective effect against CD7-expressing T-All cells both in vitro and in vivo, and could thus be expected to be further developed as a new drug for the treatment of T-ALL or other CD7-expression tumors.

RNA-binding protein NOVA1 promotes acute T-lymphocyte leukemia progression by stabilizing USP44 mRNA.

Acute T-lymphocyte leukemia (T-ALL) is a malignant tumor disease. RNA-binding protein neotumor ventral antigen-1 (NOVA1) is highly expressed in bone marrow mononuclear cells of T-ALL patients, while the role of NOVA1 in T-ALL progression remains unknown. The gain- and loss-of-function studies for NOVA1 were performed in Jurkat and CCRF-CEM cells. NOVA1 overexpression promoted cell proliferation and cell cycle progression. NOVA1 knockdown increased the apoptosis rate of T-ALL cells. Ubiquitin-specific protease 44 (USP44), a nuclear protein with deubiquitinase catalytic activity, has been reported to play an oncogene role in human T-cell leukemia. USP44 expression was positively associated with NOVA1, and RNA immunoprecipitation assay verified the binding of NOVA1 to the mRNA of USP44. USP44 knockdown partially abolished NOVA1-induced cell proliferation and inhibition of apoptosis. The in vivo xenograft experiment was performed by injection of T-ALL tumor cells into the tail vein of NOD/SCID mice. The knockdown of NOVA1 had lower tumorigenicity. NOVA1 knockdown alleviated pathological changes in lung and spleen tissues, and increased the overall survival period and the weight of T-ALL mice. Thus, NOVA1 acts as an accelerator in T-ALL, and its function might be achieved by binding to and stabilizing USP44 mRNA.

Abstract 3928: Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is hematologic tumor characterized by the diffuse infiltration of the bone marrow by malignant hematopoietic cells expressing immature T cell markers. Although T-ALL currently has better cure rates primarily due to multiagent or intensified chemotherapy, the prognosis for patients who are resistant or develop relapse to therapy remains very poor. Aberrant Notch homolog-1 (NOTCH1) signaling is a major driver of T-ALL pathogenesis as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. Gamma Secretase Inhibitors (GSIs) which effectively block the activation of oncogenic protein NOTCH1 are potential candidates for the treatment of T-ALL. However, the clinical application of GSIs is hampered by severe gastrointestinal toxicity due to the inhibition of NOTCH1 signaling in the gut. Here we demonstrate that combination therapy of GSIs and a small molecule inhibitor of the neddylation pathway circumvents the GSI-induced gut toxicity in vitro and in vivo. Genome-wide CRISPR loss-of-function screen in LS174T adenocarcinoma cells revealed neddylation pathway as a main regulator of massive goblet cell differentiation upon NOTCH1 inhibition. Genetic and pharmacologic inhibition of the neddylation pathway in LS174T cells rescued GSI-induced differentiation and cell death. Mechanistically, neddylation inhibition increases the protein stability of Hairy and enhancer of split-1 (HES1), a known regulator of absorptive and secretory cell fate decisions. Combination treatment of GSI and neddylation inhibitor in C57/BL6 mice showed a profound decrease in the number of goblet cells and maintained HES1 protein levels in the intestinal epithelium compared to GSI treatment alone. Remarkably, combined treatment of GSI and neddylation inhibitor in NOTCH1-induced T-ALL mice showed leukemia regression and improved overall survival without any associated gut toxicity. Overall, these results substantiate the potential of targeting NOTCH1 and neddylation pathway in the treatment of NOTCH1-induced T-ALL. Citation Format: Carla Bertulfo, Pablo Perez-Duran, Teresa Palomero, Adolfo Ferrando. Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3928.

A bispecific T cell engager recruits both type 1 NKT and Vγ9Vδ2-T cells for the treatment of CD1d-expressing hematological malignancies

Bispecific Tcell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory Tcells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis invitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in invivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Wnt signaling mediates oncogenic synergy between Akt and Dlx5 in T-cell lymphomagenesis by enhancing cholesterol synthesis

The Dlx5 homeobox gene was first implicated as an oncogene in a T-ALL mouse model expressing myristoylated (Myr) Akt2. Furthermore, overexpression of Dlx5 was sufficient to drive T-ALL in mice by directly activating Akt and Notch signaling. These findings implied that Akt2 cooperates with Dlx5 in T-cell lymphomagenesis. To test this hypothesis, Lck-Dlx5;Lck-MyrAkt2 transgenic mice were generated. MyrAkt2 synergized with Dlx5 to greatly accelerate and enhance the dissemination of T-lymphomagenesis. RNA-seq analysis performed on lymphomas from Lck-Dlx5;Lck-MyrAkt mice revealed upregulation of genes involved in the Wnt and cholesterol biosynthesis pathways. Combined RNA-seq and ChIP-seq analysis of lymphomas from Lck-Dlx5;Lck-MyrAkt mice demonstrated that β-catenin directly regulates genes involved in sterol regulatory element binding transcription factor 2 (Srebf2)-cholesterol synthesis. These lymphoma cells had high Lef1 levels and were highly sensitive to β-catenin and Srebf2-cholesterol synthesis inhibitors. Similarly, human T-ALL cell lines with activated NOTCH and AKT and elevated LEF1 levels were sensitive to inhibition of β-catenin and cholesterol pathways. Furthermore, LEF1 expression positively correlated with expression of genes involved in the cholesterol synthesis pathway in primary human T-ALL specimens. Together, these data suggest that targeting β-catenin and/or cholesterol biosynthesis, together with AKT, could have therapeutic efficacy in a subset of T-ALL patients.

A CXCL10/CXCR3 Driven Thymic Epithelium-Leukemia Cell Crosstalk Augments T Cell Acute Lymphoblastic Leukemia Notch1 Signalling

Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.

Surfaceome interrogation using an RNA-seq approach highlights leukemia initiating cell biomarkers in an LMO2 T cell transgenic model

The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.

Abstract A149: EµTel-Jak2 T-ALL mouse model is dependent upon Myb function and susceptible to immune checkpoint therapy

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer, predominantly in children but patients over 50 years are also affected. While high-intensity combination chemotherapy is effective in 90% of young patients, only 30–40% of adult patients with ALL will achieve long-term remission. Furthermore, the severe side effects observed in young and old patients highlight the need for alternative therapy options. To explore the role of drivers of ALL and thus the potential for other therapy options we crossed EµTEL-JAK2 mice, a mouse model of T-ALL, with MybPlt4/Plt4 mice, which express hypomorphic alleles of Myb resulting in 40% of WT Myb function. EµTEL-JAK2 x MybPlt4/Plt4 mice showed a significant prolonged survival compared to EµTEL-JAK2 Myb+/+ mice, indicating that EµTEL-JAK2 cells are dependent upon fully functional Myb. Hence, we initiated a preclinical study targeting Myb with our TetMyb DNA vaccine combined with immune checkpoint inhibitors in C57BL/6 mice bearing transplanted Eµ TEL-JAK2 T-ALL cells. Interestingly, therapeutic treatment with TetMyb DNA vaccine and anti-PD-1 antibody therapy did not enhance overall survival. However, therapeutic monotherapy with anti-CTLA-4 antibody therapy on day 2, 7, 12, 16 and 21 after EµTEL-JAK2 transplant significantly reduced leukemic cell frequency in the peripheral blood and significantly prolonged survival. Further evaluation, especially in combination with current standard of care, is needed, but our results suggest a promising therapeutic option of anti-CTLA-4 antibody therapy in T-ALL. Citation Format: Sara Roth, Shienny Sampurno, Michaela Waibel, Leonie Cluse, Lloyd A. Pereira, Ricky W. Johnstone, Robert G. Ramsay. EµTel-Jak2 T-ALL mouse model is dependent upon Myb function and susceptible to immune checkpoint therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A149.

Abstract 1128: Identification of arginine methyltransferase PRMT5 as a novel therapeutic target in T-cell acute lymphoblastic leukemia

Abstract Advances in risk-adapted cytotoxic chemotherapy, hematopoietic stem cell transplantation and supportive care have contributed to significant improvements in the survival of patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) over the past few decades. However, despite such progress, a significant percentage of both adult and pediatric leukemia patients become refractory to therapy or relapse and eventually die of disease. Hence, there remains an urgent need for the development of effective and targeted therapies for acute leukemia. Recent genetic profiling of solid and hematologic malignancies has identified epigenetic factors as a critical group of genes recurrently mutated in cancer. Additionally, epigenetic dysregulation has been shown to play an important role in the development, progression and maintenance of leukemia. Therefore, pharmacological inhibition of epigenetic factors represents a potential avenue for the development of novel epigenetic-targeted therapies. In order to identify epigenetic vulnerabilities in leukemia, we developed an epigenetic-focused shRNA screen to search for novel therapeutic targets in human leukemia cell lines both in vitro and in vivo. Specifically, T- and B-ALL cell lines were transduced with a library of shRNAs targeting 449 genes including epigenetic readers, writers and erasers and other chromatin-related factors. Selected cells were subsequently cultured in vitro and concurrently injected into mice. Engraftment of inoculated cells and disease progression were monitored through bioluminescence imaging. Amongst the universe of epigenetic regulatory proteins, the arginine methyl transferase, PRMT5, emerged as the most significantly depleted factor in both in vitro and in vivo screenings. Chemical inhibition of PRMT5 enzymatic activity effectively reduced protein symmetric dimethyl arginine methylation, altered splicing, inhibited cell growth and promoted apoptosis of both ALL and AML cell lines in vitro. In addition, inhibition of PRMT5 in vivo using patient-derived xenograft (PDX) T-ALL mouse models demonstrated diminished tumor growth and prolonged survival. Notably, quantification of peripheral blood cell numbers showed that pharmacologic PRMT5 inhibition was well tolerated and did not affect normal hematopoiesis in mice suggesting that a therapeutic window exists for anticancer drugs targeting PRMT5 in acute leukemia. Overall, our data indicates that pre-mRNA processing and in particular RNA splicing modulation may represent novel therapeutic targets in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Yunyue Wang, Hui Huang, Daniel Diolaiti, Marta Sanchez Martin, Beata Modzelewski, Lianna J. Marks, Allison R. Rainey, Ervin S. Gaviria, Maria L. Sulis, Filemon S. Dela Cruz, Adolfo A. Ferrando, Andrew L. Kung. Identification of arginine methyltransferase PRMT5 as a novel therapeutic target in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1128. doi:10.1158/1538-7445.AM2017-1128

Mine the Stability of the G2/M Checkpoint to Break Down Acute Lymphoblastic Leukemia Defenses Against Antineoplastic Drugs

Although impressive developments have been made in the treatment of Acute Lymphoblastic Leukemia (ALL) patients, the overall survival is still very poor. With the exception of novel therapeutic strategies based on monoclonal antibodies (Bi-specific T-cell engagers, BiTEs) or immunogenic cells (CART cells), the therapeutic approaches for adult ALL patients are still base on non-selective chemotherapy or on tyrosine kinase inhibitors (TKIs) for the patients harboring the BCR-ABL1 fusion transcript. In addition a large percentage of initial successfully treated patients frequently develop relapses. Thus there is a need to improve the efficacy of conventional therapies, in particular those related to TKIs and to DNA damaging agents, in order to reduce the off-target toxicity and avoid relapses. In the present study we evaluated the in vitro, ex vivo and in vivo efficacy of MK-1775, a specific Wee1 inhibitor, in single agent and in combination with different therapeutic agents normally used for the treatment of B-/T-ALL. We firstly started by evaluated the efficacy of the compound in single agent on a panel of human B and T ALL cell lines (n=8) and on primary cells isolated from the bone marrow of adult B-ALL patients (n=8). The inhibition of Wee1 deeply reduced the cell viability and the proliferation rate, induced the apoptosis and increased the DNA damages of both leukemic cell lines and primary cells. Further cell-cycle analysis showed that in leukemic cell lines the treatment increased the number of cell in late S and G2/M phase. Light microscopy analyses, looking for nuclei morphology, confirmed that MK-1775 increased the number of mitotic cells but it interfered with normal mitotic division (induction of aberrant mitosis as showed by the increment of DNA bridges and micro-nuclei). The effects of the compound on the cell cycle profile and on the G2/M checkpoint were confirmed also in immunoblotting analyses, by the increment of phospho-HH3(ser10) and of Myt1 (mitotic isoform), and by gene expression analysis looking to specific genes involved in the G2/M checkpoints (PrimePcr DNA damage assay, Biorad). In particular genes like GADD45A and CCNB1/CCNB2 were significantly up-regulated between treated and untreated samples. Finally using a T-ALL mouse model we evaluated the effect of MK-1775 in single agent. Although no significative differences were seen between treated and un-treated samples, due to a very aggressive phenotype of the disease (all animal died after only 18 days from the engraftment), molecular analyses confirmed that the treatment induced DNA damages (increase of H2A.X and p-Chk1 ser317) and inhibited Wee1 functionality (reduction of pCDC2) on leukemic blasts isolated from both spleens and bone marrows. To evaluate if the inhibition of the G2/M checkpoint could sensitize leukemic cells to the toxicity of antineoplastic drugs, Philadelphia-negative ALL cell lines and primary leukemic cells (n=9) where treated with increasing concentration of MK-1775 and increasing concentration of the nucleotide analogue, clofarabine. Statistical analyses (Combination index value) confirmed the synergy of the combination in the reduction of the cell viability, in the inhibition of the proliferation and in the induction of the apoptosis. Similar results were seen on Philadelphia-positive ALL cell lines and primary cells (n=3) combining the MK-1775 with the TKI, bosutinib. The simultaneously inhibition of the Wee1 and the BCR-ABL downstream pathway resulted in a synergic inhibition of the cell viability, reduction of the proliferation and induction of apoptosis. In our opinion the pre-clinical results of this study are the basis for a future clinical evaluation of MK-1775 for the treatment of ALL patients.Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. DisclosuresMartinelli:Novartis: Speakers Bureau; BMS: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau.

Abstract LB-181: KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with the highest incidence of relapse of any pediatric ALL. A minimal two-hit model of leukemogenesis suggests that an initial genetic driver transforms hematopoietic progenitor cells into LICs, whereas a secondary genetic alteration would endow LICs with proliferative and survival advantages. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate onset of leukemia and worsen disease progression are largely unknown. Here, we show that low levels of the transcription factor KLF4 in children with T-ALL were associated with methylation of its promoter. Consistent with a tumor suppressor function, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL in mice by enhancing the G1-to-S transition and promoting the expansion of leukemia-initiating cells that are responsible for chemoresistance and relapses. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7, and thus loss of KLF4 activates MAP2K7 and downstream effector JNK both in murine model of T-ALL and lymphoblasts from pediatric patients with T-ALL. Furthermore, pharmacological inhibition of JNK reduced leukemia burden in a xenograft model of human T-ALL and small molecule inhibitors exhibited anti-leukemic properties in patient-derived xenograft cells. In summary, our findings demonstrate a novel tumor suppressor function of KLF4 in a T-ALL mouse model and in pediatric T-ALL and support a model of leukemia inhibition by repression of the stress kinase MAP2K7 and its downstream targets JNK, c-JUN, and ATF2. In addition, our study provides proof-of-principle pre-clinical data supporting JNK inhibition as a potential targeted therapy for T-ALL and prompts future studies in high-risk T-ALL patients with refractory and relapsed disease. Citation Format: Ye Shen, Chun Shik Park, Koramit Suppipat, Toni-Ann Mistretta, Monica Puppi, Terzah Horton, Karen Rabin, Daniel Lacorazza. KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway. [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-181.

Effect of PDK1 on Notch1-Induced Mouse T-cell Acute Lymphoblastic Leukemia

To explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1. Cell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR. Notch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully. Compared to T-ALL control mouse model, PDK1 knock-out mice showed a significant longer survival time (P<0.01). There was no difference of cell cycle between control and PDK1 knock-out mice, and the apoptosis rate of leukemic cells in PDK1 knock-out mice was higher than that of control mice (P<0.001). PDK1 knock-out resulted in decreased expression of tumor-related genes and transcription factors, such as c-Myc and NF-κB (P<0.01), and increased expression level of P53 (P<0.01). PDK1 knock-out can inhibit the development of T-ALL, and its mechanism may be the leukemia progression inhibited by regulating the apoptosis and expression of multiple related genes and transcription factors.

Abstract B19: Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.

Abstract Acute lymphoblastic leukemia is the most common hematological malignancy in pediatric patients, and disease relapse is the leading cause of cancer-associated death in children. Despite steadily improved outcomes in patients with newly diagnosed disease, little progress has been made to treat relapse leukemia and to reduce the incidence of relapse by increasing the cure rates of frontline therapy. Targeted therapy is currently not available for T-ALL and the development of novel agents requires a better understanding of how leukemia-initiating cells (LIC) are maintained. In contrast to normal blood cells, we found that the transcription factor KLF4 is significantly downregulated in pediatric T-ALL. Next-generation bisulfite sequencing revealed that the KLF4 promoter is hypermethylated in lymphoblasts from T-ALL patients but not in normal bone marrow cells, T cells, and pediatric B-ALL. Deletion of the Klf4-floxed gene with Vav-iCre transgene and transplantation of Klf4Δ/Δ bone marrow cells transduced with the Notch1-L1601P-ΔP mutant resulted in a significant acceleration of T-ALL due to a 9-fold expansion of LIC and increased G1-S transition in leukemic T cells. A combined analysis of global gene expression and genome-wide binding revealed that KLF4 directly represses the dual specificity mitogen-activated protein kinase 7 (Map2k7) and thus Klf4Δ/Δ T-ALL mice exhibit increased Map2k7 expression. Most remarkably, T-ALL cells from both mouse model and pediatric patients showed elevated levels of total and phosphorylated Map2k7 and subsequent activation of downstream targets JNK, c-Jun, and ATF2. Finally, we tested several JNK inhibitors in T-ALL cell lines, primary patient samples, and pre-clinical xenograft models of T-ALL and confirmed that JNK inhibition blocks ATF2 activation and controls expansion of leukemia cells, validating the Map2k7/Jnk pathway as a selective target for T-ALL therapy. Thus, KLF4 emerges as a novel tumor suppressor in T-ALL and Jnk inhibition as a novel therapeutic approach for adjunctive therapy in T-ALL patients with refractory or relapse disease. Citation Format: Ye Shen, Koramit Suppipat, Chun Shik Park, Toni-Ann Mistretta, Terzah Horton, Karen Rabin, Daniel Lacorazza. Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B19.

Altered mesenchymal niche cells impede generation of normal hematopoietic progenitor cells in leukemic bone marrow.

Degeneration of normal hematopoietic cells is a shared feature of malignant diseases in the hematopoietic system. Previous studies have shown the exhaustion of hematopoietic progenitor cells (HPCs) in leukemic marrow, whereas hematopoietic stem cells (HSCs) remain functional upon relocation to non-leukemic marrow. However, the underlying cellular mechanisms, especially the specific niche components that are responsible for the degeneration of HPCs, are unknown. In this study, we focused on murine bone mesenchymal stem cells (MSCs) and their supporting function for normal hematopoietic cells in Notch1-induced acute T-cell lymphocytic leukemia (T-ALL) mice. We demonstrate that the proliferative capability and differentiation potential of T-ALL MSCs were impaired due to accelerated cellular senescence. RNA-seq analysis revealed significant transcriptional alterations in leukemic MSCs. After co-cultured with the MSCs from T-ALL mice, a specific inhibitory effect on HPCs was defined, whereas in vivo repopulating potential of normal HSCs was not compromised. Furthermore, osteoprotegerin was identified as a cytokine to improve the function of T-ALL MSCs and to enhance normal HPC output via the p38/ERK pathway. Therefore, this study reveals a novel cellular mechanism underlying the inhibition of HPC generation in T-ALL. Leukemic MSCs may serve as a cellular target for improving normal hematopoietic regeneration therapeutically.

Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca2+ response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

The Supportive Role Of Mesenchymal Stem Cells On Normal Hematopoietic Stem Cells Is Impaired In Leukemic Bone Marrow

Mesenchymal stem cells (MSCs) have been shown to be able to form a niche that maintains hematopoietic stem cells (HSCs). However, how the phonotypical characteristics and biological functions of MSCs are affected and especially whether their supporting role on normal HSC is impaired in leukemic microenvironment, are not well defined. Here, we have examined the changes of biological characteristics of mouse MSCs and in particular their supporting function on normal HSCs in Notch1-induced acute T cell lymphocytic leukemia (T-ALL) mice (Hu et al, Blood 2009; 114:3783-3792). MSCs were sorted based on the phenotypic markers, PDGFRα+Sca-1+CD45-TER119- (PαS) (Houlihan et al, Nat Protoc 2012) from control or T-ALL mice and flow cytometric analysis was conducted at different time points during leukemia development. Our results showed there was no significant difference between T-ALL and control mice regarding the absolute number and percentage of PαS MSCs in total bone marrow mono-nuclear cells (BMMNCs), cell cycling status and the percentage of apoptosis of freshly sorted PαS MSCs. Moreover, PαS MSCs from both normal and T-ALL leukemic marrow had similar morphology (spindle and polymorphic shaped), and uniformly expressed known cell surface markers for cultured mouse MSCs (CD29, CD44, Sca-1). However, the number of T-ALL PαS MSCs colony forming unit-fibroblastic (CFU-F) formation was 3-fold lower than control CFU-F (p<0.001). Levels of mRNA expression of genes associated with adipogenic (adipsin, mLP, PPARγ), osteogenic (Bmp4, Sp7, Bglap) and chondrogenic (collagen II, collagen X, aggrecan) differentiation in PαS MSCs were significantly decreased in T-ALL mice compared with control mice (p<0.05). These results suggest that the proliferation potential and the differentiation potential of MSC were decreased in the leukemic environment. To investigate whether the decrease in the proliferation of T-ALL PαS MSCs was associated with a change in cellular senescence, β-galactosidase activity and quantitative RT-PCR analysis of genes associated with senescence were performed. Our finding showed a significant increase in the number of β-galactosidase–positive cells (control/T-ALL; 3.11±0.20%/3.99±0.08%, p<0.05) and in the mRNA expression level of senescence-related gene p16 in PαS MSCs T-ALL mice compared with control mice (control/T-ALL; 1.00±0.03/1.57±0.17, p<0.05). These results imply that p16 plays an important role in PαS MSCs senescence in T-ALL microenvironment. To determine whether the supporting function of T-ALL PαS MSCs on normal HSCs proliferation is impaired, the cobble-stone area forming cell (CAFC) assay, an in vitro surrogate for HSCs, was applied. Normal control or T-ALL PαS MSCs were co-cultured with the same pool of normal HSCs. After 5 weeks, co-cultures containing normal PαS MSC formed multiple large CAFCs. On the contrast, T-ALL PαS MSCs supported 7-fold less hypocellular CAFCs (p<0.05). This data indicate that T-ALL PαS MSCs have a reduced ability to support normal HSCs proliferation in vitro. To further validate the change of PαS MSCs' supporting function on normal HSCs proliferation in leukemic microenvironment observed in vitro, in vivo co-transplantation study were performed. Our preliminary results further indicated that the supporting function of MSCs on normal hematopoiesis in the leukemic microenvironment was compromised. In conclusion, dysfunction of MSC, an important component of HSC microenvironment, may play a crucial role in the suppression of HSC during leukemia development. Improving the function of MSC may serve as a new strategy to enhance normal hematopoiesis in leukemic marrow. Disclosures:No relevant conflicts of interest to declare.

Abstract B12: Co-inhibition of PI3K and Myc pathways is synthetic lethal to PTEN null T-ALL leukemia-initiating cells

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy associated with poor prognosis and significant risk of relapse. Loss of PTEN function due to mutations or deletions is common in primary T-ALL samples and is associated with resistance to therapeutic agents. We previously developed a VEC-Cre+;Ptenloxp/loxp (Pten null) T-ALL model to investigate the molecular and cellular mechanisms underlying T-ALL pathogenesis and resistance to therapy. Pten-null T-ALL develops after acquisition of a T cell receptor α/δ-c-myc translocation that causes c-Myc over-expression, and the leukemia-initiating cells, or leukemia stem cells (LSCs), are enriched in a c-kitmidCD3+ subpopulation. The goal of our current study is to develop improved therapeutic approaches to target LSCs and eliminate T-ALL. We hypothesize that small molecule inhibitors that target molecular lesions and pathways required for the development and survival of Pten null LSCs and T-ALL may be potent in eliminating LSCs and abolishing T-ALL. Rapamycin, an inhibitor of the PI3K downstream kinase mTOR, can suppress leukemia development in Pten null pre-leukemic mice, but is insufficient to eliminate LSCs and T-ALL. Therefore, we are investigating the therapeutic utility of combinational therapies that co-target multiple deregulated pathways that are critical for LSC formation and T-ALL survival. We found that combination treatment of Pten null T-ALL mice with rapamycin and VX-680, an Aurora kinase inhibitor shown to have synthetic lethality with Myc over-expression, causes robust elimination of leukemia blasts and significantly diminishes the LSC population. Functional studies for leukemia-initiating-cell activity using transplantation of treated bone marrow into NOD-SCID-IL2rγ-/-(NSG) recipients showed that dual treatment reduced leukemia-initiating-cell activity in bone marrow by at least 100-fold in comparison to single agent alone. Importantly, a large portion of Pten null T-ALL LSCs are actively cycling (~50% in S-phase), which may contribute to the mechanism underlying the efficacy of VX-680. We have developed a T-ALL in vitro culture system derived from primary Pten null T-ALL mice to conduct more detailed evaluation of the cellular and biochemical effects of rapamycin and VX-680, perform mechanistic analysis, and screen additional cell cycle-directed and Myc-targeted agents. We found that Pten null T-ALL is highly sensitive to killing by the CDK1 inhibitor purvalanol and JQ1, a bromodomain inhibitor reported to down-regulate c-myc expression that is suitable for use in vivo. Rapamycin and JQ1 combination treatment of Pten null T-ALL mice causes a significant reduction in both the leukemia blast and LSC populations, similar to the effects of rapamycin and VX-680 combination therapy. Our results highlight the importance of understanding the mechanisms underlying T-ALL development and survival and show potent synthetic lethality of PI3K and Myc pathway co-inhibition in PTEN null T-ALL LSCs. Importantly, such targeted combination therapy may offer an improved therapeutic approach to treat human T-ALL. Citation Format: Suzanne Schubbert, Anjelica Cardenas, Christopher Wei, Lingda Ma, Consuelo Garcia, Wei Guo, Hong Wu. Co-inhibition of PI3K and Myc pathways is synthetic lethal to PTEN null T-ALL leukemia-initiating cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B12.

Notch1 signaling is involved in regulating Foxp3 expression in T-ALL

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy. Immune tolerance induced by CD4+CD25+ regulatory T cells (Tregs) with high expression of Foxp3 is an important hypothesis for poor therapy response. Notch1 signaling is thought to be involved in the pathogenesis of this disease. Crosstalk between Notch and Foxp3+Tregs induced immune tolerance is unknown in T-ALL. We studied Foxp3 and Notch1 expression in vivo and in vitro, and analyzed the biological characteristics of T-ALL cell line systematically after Notch inhibition and explored the crosstalk between Notch signaling and Foxp3 expression.MethodsIn vivo, we established T-ALL murine model by Jurkat cells transplantation to severe combined immunodeficiency (SCID) mice. Notch1 and Foxp3 expression was detected. In vitro, we used γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) to block Notch1 signaling in Jurkat cells. Notch1, Hes-1 and Foxp3 genes and protein expression were detected by PCR and western blotting, respectively. The proliferation pattern, cell cycle and viability of Jurkat cells after DAPT treatment were studied. Protein expression of Notch1 target genes including NF-κB, p-ERK1/2 and STAT1 were determined.ResultsWe show that engraftment of Jurkat cells in SCID mice occurred in 8 of 10 samples (80%), producing disseminated human neoplastic lymphocytes in PB, bone marrow or infiltrated organs. Notch1 and Foxp3 expression were higher in T-ALL mice than normal mice. In vitro, Jurkat cells expressed Notch1 and more Foxp3 than normal peripheral blood mononuclear cells (PBMCs) in both mRNA and protein levels. Blocking Notch1 signal by DAPT inhibited the proliferation of Jurkat cells and induced G0/G1 phase cell cycle arrest and apoptosis. Foxp3 as well as p-ERK1/2, STAT1 and NF-κB expression was down regulated after DAPT treatment.ConclusionsThese findings indicate that regulation of Foxp3 expression does involve Notch signaling, and they may cooperatively regulate T cell proliferation in T-ALL.

Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Abstract 1216During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model.Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory.In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures:No relevant conflicts of interest to declare.