T-cell Acute Lymphoblastic Leukemia Xenografts Research Articles

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

Canonical BAF complex regulates the oncogenic program in human T-cell acute lymphoblastic leukemia

AbstractAcute leukemia cells require bone marrow microenvironments, known as niches, which provide leukemic cells with niche factors that are essential for leukemic cell survival and/or proliferation. However, it remains unclear how the dynamics of the leukemic cell–niche interaction are regulated. Using a genome-wide CRISPR screen, we discovered that canonical BRG1/BRM-associated factor (cBAF), a variant of the switch/sucrose nonfermenting chromatin remodeling complex, regulates the migratory response of human T-cell acute lymphoblastic leukemia (T-ALL) cells to a niche factor CXCL12. Mechanistically, cBAF maintains chromatin accessibility and allows RUNX1 to bind to CXCR4 enhancer regions. cBAF inhibition evicts RUNX1 from the genome, resulting in CXCR4 downregulation and impaired migration activity. In addition, cBAF maintains chromatin accessibility preferentially at RUNX1 binding sites, ensuring RUNX1 binding at these sites, and is required for expression of RUNX1-regulated genes, such as CDK6; therefore, cBAF inhibition negatively impacts cell proliferation and profoundly induces apoptosis. This anticancer effect was also confirmed using T-ALL xenograft models, suggesting cBAF as a promising therapeutic target. Thus, we provide novel evidence that cBAF regulates the RUNX1-driven leukemic program and governs migration activity toward CXCL12 and cell-autonomous growth in human T-ALL.

Idasanutlin and navitoclax induce synergistic apoptotic cell death in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.

A CH2CH3 hinge region enhances the cytotoxicity of anti-CD5 CAR-T cells targeting T cell acute lymphoblastic leukemia

Chimeric antigen receptor T cell (CAR-T) therapies show considerable clinical efficacy in patients with B cell malignancies, but their efficacy is limited in patients with T cell acute lymphoblastic leukemia (T-ALL). CD5 is expressed on ∼85 % of malignant T cells, and CD5-targeting CAR-T cells can exhibit potent antitumor activity against T-ALL. However, optimization of CAR costimulatory endo-, hinge, and transmembrane domains could further increase their expansion and persistence, thereby enhancing their efficacy following exposure to tumor cells. Here we designed CD5-specific CARs with different molecular structures to generate CAR-T cells and investigated their anti-tumor efficacy in vitro and in vivo. CD5 CARs with a 4-1BB costimulatory domain (BB.z) or a CD28 costimulatory domain (28.z) exhibited specific cytotoxicity against CD5+ malignant cells in vitro. However, both failed to prolong the survival of T-ALL xenograft mice. Subsequently, we substituted the 28.z CAR hinge region with CH2CH3, which enhanced the ability of CH2CH3-CD5 CAR-T cells to specifically eradicate T-ALL cells in vitro and in vivo. Furthermore, patient-derived CH2CH3-CD5 CAR-T cells were generated which showed a marked killing effect of CD5-positive acute T-ALL cells in vitro. The anti-tumor activity of CD5 CAR-T cells with a CD28 co-stimulation domain and CH2CH3 hinge region was superior to those with BB.z and 28.z domains. These preclinical data provided new insights into the factors dictating efficacy in T-ALL treatment with CAR-T cells and hold promise for clinical translation.

Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

BackgroundPatients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous...

YBX1 as an oncogenic factor in T-cell acute lymphoblastic leukemia.

Y-box-binding protein 1 (YBX1), a member of the RNA-binding protein family, is a critical regulator of cell survival in various solid tumors and acute myeloid leukemia. However, the function of YBX1 in T-cell acute lymphoblastic leukemia (T-ALL) remains elusive. Here, we found that YBX1 was upregulated in patients with T-ALL, T-ALL cell lines, and NOTCH1-induced T-ALL mice. Furthermore, depletion of YBX1 dramatically reduced cell proliferation, induced cell apoptosis, and induced G0/G1 phase arrest invitro. Moreover, YBX1 depletion significantly decreased the leukemia burden in the human T-ALL xenograft and NOTCH1-induced T-ALL mice model invivo. Mechanistically, downregulation of YBX1 markedly inhibited the expression of total AKT serine/threonine kinase (AKT), p-AKT, total extracellular signal-regulated kinase (ERK), and p-ERK in T-ALL cells. Taken together, our results uncovered a critical role of YBX1 in the leukemogenesis of T-ALL, which may have great potential as a biomarker and therapeutic target in T-ALL.

S1P-S1PR3-RAS promotes the progression of S1PR3hi TAL1+ T-cell acute lymphoblastic leukemia that can be effectively inhibited by an S1PR3 antagonist.

TAL1+ T-cell acute lymphoblastic leukemia (T-ALL) is a distinct subtype of leukemia with poor outcomes. Through the cooperation of co-activators, including RUNX1, GATA3, and MYB, the TAL1 oncoprotein extends the immature thymocytes with autonomy and plays an important role in the development of T-ALL. However, this process is not yet well understood. Here, by investigating the transcriptome and prognosis of T-ALL from multiple cohorts, we found that S1PR3 was highly expressed in a subset of TAL1+ T-ALL (S1PR3hi TAL1+ T-ALL), which showed poor outcomes. Through pharmacological and genetic methods, we identified a specific survival-supporting role of S1P-S1PR3 in TAL1+ T-ALL cells. In T-ALL cells, TAL1-RUNX1 up-regulated the expression of S1PR3 by binding to the enhancer region of S1PR3 gene. With hyperactivated S1P-S1PR3, T-ALL cells grew rapidly, partly by activating the KRAS signal. Finally, we assessed S1PR3 inhibitor TY-52156 in T-ALL patient-derived xenografts (PDXs) mouse model. We found that TY-52156 attenuated leukemia progression efficiently and extended the lifespan of S1PR3hi TAL1+ T-ALL xenografts. Our findings demonstrate that S1PR3 plays an important oncogenic role in S1PR3hi TAL1+ T-ALL and may serve as a promising therapeutic target.

Anlotinib exerts an anti-T-cell acute lymphoblastic leukemia effect in vitro and in vivo

BackgroundDespite some progress having been made regarding the treatment of T-cell acute lymphoblastic leukemia (T-ALL), the prognosis of T-ALL, particularly adult T-ALL, is still poor. Identifying novel, effective anti-T-ALL drugs is of great significance. Anlotinib, an oral tyrosine kinase inhibitor currently utilized in the treatment of lung cancer, exhibited a promising anti-T-ALL effect. A comprehensive study should therefore be conducted to explore both the in vitro as well as in vivo mechanisms of the anti-T-ALL effects of anlotinib. MethodsCCK8 assays and flow cytometry were employed to investigate the viability, cell cycle distribution, and apoptosis of T-ALL cell lines when treated with anlotinib. T-ALL xenograft mouse models were established to examine the in vivo antileukemic effects of anlotinib. Cellular and molecular analysis of T-ALL were conducted to define the underlying mechanisms. ResultsIn vitro, anlotinib significantly inhibited the viability, induced G2/M phase arrest and apoptosis in T-ALL cell lines in a concentration-dependent pattern. In vivo, anlotinib also demonstrated a strong anti-tumor effect at doses that are well-tolerated. Interestingly, anlotinib could decrease the protein levels of the intracellular domains of NOTCH1 (ICN1) and c-Myc, two important targets for T-ALL. Mechanistically, anlotinib-induced c-Myc reduction was associated with proteasome-mediated degradation, while the ICN1 reduction was not due to protein degradation or transcriptional repression. ConclusionsThe present study showed that anlotinib may be a promising anti-T-ALL candidate drug, and simultaneous reduction of the protein levels of both ICN1 and c-Myc may contribute to the anti-T-ALL efficacy of anlotinib.

WDR54 exerts oncogenic roles in T-cell acute lymphoblastic leukemia.

WDR54 has been recently identified as a novel oncogene in colorectal and bladder cancers. However, the expression and function of WDR54 in T-cell acute lymphoblastic leukemia (T-ALL) were not reported. In this study, we investigated the expression of WDR54 in T-ALL, as well as its function in T-ALL pathogenesis using cell lines and T-ALL xenograft. Bioinformatics analysis indicated high mRNA expression of WDR54 in T-ALL. We further confirmed that the expression of WDR54 was significantly elevated in T-ALL. Depletion of WDR54 dramatically inhibited cell viability and induced apoptosis and cell cycle arrest at S phase in T-ALL cells in vitro. Moreover, knockdown of WDR54 impeded the process of leukemogenesis in a Jurkat xenograft model in vivo. Mechanistically, the expression of PDPK1, phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), Bcl-2 and Bcl-xL were downregulated, while cleaved caspase-3 and cleaved caspase-9 were upregulated in T-ALL cells with WDR54 knockdown. Additionally, RNA-seq analysis indicated that WDR54 might regulate the expression of some oncogenic genes involved in multiple signaling pathways. Taken together, these findings suggest that WDR54 may be involved in the pathogenesis of T-ALL and serve as a potential therapeutic target for the treatment of T-ALL.

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.

Therapeutic Targeting of MERTK and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical Development of Non-Edited CD7 CAR-Modified Invariant NKT Cell Therapy for T-Cell Malignancies and Acute Myeloid Leukemia

The major hurdle in developing chimeric antigen receptor T (CAR-T) cell therapy for T-cell malignancies is CAR-T cell fratricide. This refers to the self-killing of the CAR-T cells due to shared expression of targetable antigens (e.g., CD7) between CAR-T and malignant target T cells. This CAR-T fratricide creates difficulties in producing sufficient numbers of CAR-T cells for infusion and can limit survival of the cells in vivo. Nuclease (e.g., CRISPR)-mediated genomic disruption of CD7 or T cell receptor alpha chain expression provides an alternative approach to enabling expansion of CD7 CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based genome editing remain unclear. Thus, other approaches to development of allogeneic, non-edited CD7 CAR-T cell therapy for relapsed and refractory T-cell malignancies is warranted. We unexpectedly found that human invariant natural killer T (iNKT) cells in normal blood donors displayed on average approximately 56% CD7 positivity (range 38 - 74%, n = 9 donors). Therefore, we hypothesize that CD7- subsets in iNKT cells expressing CD7 CAR can partially avoid fratricide without nuclease-mediated genome editing to delete CD7 and be expanded for adoptive cell therapy of CD7+ malignancies. By leveraging our proprietary 6000-10000-fold iNKT cell expansion platform, lentiviral transduced CD7 CAR iNKT (CD7 CAR-iNKT) cells can be expanded to clinical scale (n=2) (Fig. 1A). Based on CD19 CAR- and CD7 CAR-iNKT expansion rate (n=5), 100-500 doses (assuming 1-3 infusions per patient) of CAR-iNKT cells can be manufactured from a single donor-derived leukocyte concentrate. CD7 CAR-iNKT cells (n=5, 64-98% CAR+) specifically killed luciferase-expressing CD7+ T-cell acute lymphoblastic leukemia (T-ALL) (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1) at E/T ratios ranging from 20:1 to 2.2:1 compared to mock iNKT cells and CD19 CAR-iNKT cells (p < 0.0001). Control CD19 CAR-iNKT cells (n=3) specifically killed CD19+ B-cell tumor cells but not CD19- T-ALL and AML cells (p < 0.0001). Mock iNKT cells showed minimal cytotoxicity to all target cells except for CD1d+ Jurkat. CD7 CAR-iNKT cells (n=5) also produced significant amounts of IFN-γ in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1, Kasumi-6) compared to mock (n=5, p < 0.001) and CD19 CAR-iNKT cells (p < 0.05 except for Jurkat, p < 0.01). CD19 CAR-iNKT cells (n=3) produced significant amounts of IFN-γ to CD19+ B-cell leukemia and lymphoma cell lines (Nalm6, Daudi, Raji) compared to mock and CD7 CAR-iNKT cells (p < 0.0001). As expected, CD7 CAR-iNKT cells significantly eliminated almost all CD7+ cells (mean ± SD 0.4% ± 0.1, n=4) compared to mock (61.6% ± 24.4, n=4) and CD19 or ROR1 CAR-iNKT cells (69.9% ± 24.4, n=3) (p < 0.0001). Furthermore, expanded CD7 CAR-iNKT cells were >95% CD3+TCRVα24-Jα18+ and specifically responded to a-galactosylceramide or 7DW8-5 lipid antigens presented by CD1d molecules. The anti-leukemic effect of CD7 CAR-modified iNKT cells was assessed in HSB2 T-ALL and KG1 AML xenograft models (n=5 per group). Three i.v. infusions of thawed CD7 CAR-iNKT cells (5x106 cells/mouse) at days 0, 3, and 6 into leukemia (5x105/mouse, i.v. at day -2)-bearing mice significantly suppressed T-ALL growth by bioluminescent imaging analysis at days 3 (p < 0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024) and 17 (p < 0.0001) compared to PBS and mock controls (Fig. 1B). A significant survival advantage was observed after CD7 CAR-iNKT cell therapy compared to PBS and mock controls (p = 0.0009, Fig. 1C). Two i.v. infusions of thawed CD7 CAR-iNKT cells into pre-established AML mice significantly suppressed AML growth at days 3 (p = 0.0096), 6 (p < 0.0001), 10 (p = 0.0026), 17 (p < 0.0001), 24 (p < 0.0001), 31 (p < 0.0001), 38 (p < 0.0001) and 45 (p = 0.0003) compared to mock control (Fig. 1D). CD7 CAR-iNKT cell therapy also significantly prolonged the animal survival (p = 0.0069, Fig. 1E). In conclusion, these results demonstrate that CD7 CAR-iNKT cells without genome editing can be manufactured to therapeutic doses and mount anti-CD7+ tumor responses in vitro and in vivo. Our CD7 CAR-iNKT cells can be used as allogeneic, off-the-shelf products to treat all subtypes of T-ALL and 30% of CD7+ AML patients. In addition, our CD7 CAR-iNKT cells are likely more effective in 70% CD7+CD1d+ T-ALL patients as they provide dual target specificity and reduce the likelihood of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of the m6A reader IGF2BP2 as a strategy against T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant leukemia with extremely limited treatment for relapsed patients. N6‐methyladenosine (m6A) reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) participates in the initiation and growth of cancers by communicating with various targets. Here, we found IGF2BP2 was highly expressed in T-ALL. Gain and loss of IGF2BP2 demonstrated IGF2BP2 was essential for T-ALL cell proliferation in vitro and loss of IGF2BP2 prolonged animal survival in a human T-ALL xenograft model. Mechanistically, IGF2BP2 directly bound to T-ALL oncogene NOTCH1 via an m6A dependent manner. Furthermore, we identified a small-molecule IGF2BP2 inhibitor JX5 and treatment of T-ALL with JX5 showed similar functions as knockdown of IGF2BP2. These findings not only shed light on the role of IGF2BP2 in T-ALL, but also provide an alternative γ‑Secretase inhibitors (GSI) therapy to treat T-ALL.

A Novel Bispecific T-Cell Engager (CD1a x CD3ε) BTCE Is Effective against Cortical-Derived T Cell Acute Lymphoblastic Leukemia (T-ALL) Cells.

Simple SummaryT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and still orphan hematologic malignancy. No effective immunotherapeutic strategies are presently available for this poor prognosis disease. We here report the development and the preclinical evaluation of a novel bispecific T-cell engager (BTCE) that simultaneously targets CD1a and CD3ε (CD1a x CD3ε), therefore recruiting T cells against T-ALL cells. We demonstrate that this CD1a x CD3ε BTCE induces activation, proliferation, and cytokine release by T cells in co-cultures with CD1a expressing T-ALL cells, resulting in a concentration-dependent killing of leukemic cells in vitro. Moreover, CD1a x CD3ε BTCE inhibits the in vivo growth of human T-ALL xenografts and improves survival of immunocompromised mice reconstituted with human PBMC from healthy donors. We believe that this BTCE is suitable for clinical development for the treatment of CD1a-expressing T-ALL patients.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy burdened by poor prognosis. While huge progress of immunotherapy has recently improved the outcome of B-cell malignancies, the lack of tumor-restricted T-cell antigens still hampers its progress in T-ALL. Therefore, innovative immunotherapeutic agents are eagerly awaited. To this end, we generated a novel asymmetric (2 + 1) bispecific T-cell engager (BTCE) targeting CD1a and CD3ε (CD1a x CD3ε) starting from the development of a novel mAb named UMG2. UMG2 mAb reacts against CD1a, a glycoprotein highly expressed by cortical T-ALL cells. Importantly, no UMG2 binding was found on normal T-cells. CD1a x CD3ε induced high T-cell mediated cytotoxicity against CD1a+ T-ALL cells in vitro, as demonstrated by the concentration-dependent increase of T-cell proliferation, degranulation, induction of cell surface activation markers, and secretion of pro-inflammatory cytokines. Most importantly, in a PBMC-reconstituted NGS mouse model bearing human T-ALL, CD1a x CD3ε significantly inhibited the growth of human T-ALL xenografts, translating into a significant survival advantage of treated animals. In conclusion, CD1a x CD3ε is a novel BTCE highly active against CD1a-expressing cortical-derived T-ALL cells suitable for clinical development as an effective therapeutic option for this rare and aggressive disease.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

Combining daratumumab with CD47 blockade prolongs survival in preclinical models of pediatric T-ALL

AbstractAcute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2–like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease–like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.

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.