Lymphoblastic Leukemia Cell Line Research Articles

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.

Novel CD20 Mutations As a Mechanism of Resistance to CD20-CD3 Targeted Therapies in Non-Hodgkin's Lymphoma

Non-Hodgkin's Lymphoma (NHL) is one of the most common blood cancer types in the world. CD20 is a B cell restricted lineage marker that is a well-established target for the treatment of NHL and anti-CD20 agents are included as in standard of care regimens. Given the success of these targeted therapies, strategies to further enhance therapeutic activity are being explored, including the CD20-CD3 bispecific molecule, mosunetuzumab, which is approved for treatment of relapsed and refractory Follicular Lymphoma in adults who have received >2 lines of treatment. Mosunetuzumab acts by redirecting CD3+ T cells to engage and eliminate CD20-expressing B cells. As with other targeted therapies, mutations and loss of target expression have been described following mosunetuzumab therapy [1]. Here we describe the impact of novel mutations identified in patient biopsy samples from the mosunetuzumab monotherapy trial, GO29781 that were located within the extracellular and transmembrane domains of CD20 on protein expression, localization and T-cell mediated killing. NHL cell lines, SU-DHL-16 and MAVER-1, were engineered using CRISPR-Cas9 system to create CD20 knock out (KO) cell lines. Wild-type (WT) or mutant CD20 were exogenously re-expressed in the CD20 KO cell lines and in the CD20 negative acute lymphoblastic leukemia cell line, REH, to create isogenic cell lines for further analysis. Complete CD20 loss was observed in cells expressing CD20 P160fs (frameshift) and Q187* (truncating) mutations. CD20 missense mutations located in the extracellular domain (C167G, K175E) expressed CD20 protein levels comparable to CD20 WT cells and membrane localization was confirmed by flow cytometry and immunofluorescence microscopy. To test the functional effect of CD20 mutations, in vitro co-culture cell killing assays were carried out using healthy donor CD8+ T cells, isogenic cell lines (3:1 effector to tumor cell ratio) and a proof-of-concept CD20-CD3 bispecific antibody. Differences in T-cell mediated killing were observed across mutation types. CD20 WT cell lines demonstrated 95-99% cell killing following 48 hour treatment while the CD20 KO, frameshift and truncating mutations all showed negligible T-cell mediated killing (~10-17% cell death). Cells expressing the missense mutations in the extracellular domain showed resistance to T-cell mediated killing (~30-45% cell death) compared to CD20 WT cells. T-cell activation was assessed by CD69 (early) and CD25 (late) surface markers. In all co-cultures, the level of CD69+ T cells was equivalent, while the CD69+CD25+ CD8 T cells were only significantly increased in the CD20 WT co-cultures, consistent with the high level of T-cell mediating killing. We have confirmed that these mutations can play a role in the development of resistance through both loss of target protein expression and interference within the anti-CD20 binding site. These studies help to characterize mechanisms of resistance to mosunetuzumab that could be extended to other anti-CD20 targeted agents. [1] Schuster et al. Journal of Clinical Oncology 2022, 40 (16), 7526.

Dependency of B-Cell Acute Lymphoblastic Leukemia and Multiple Myeloma Cell Lines on MEN1 Extends beyond MEN1-KMT2A Interaction.

Menin/MEN1 is a scaffold protein that participates in proliferation, regulation of gene transcription, DNA damage repair, and signal transduction. In hematological malignancies harboring the KMT2A/MLL1 (MLLr) chromosomal rearrangements, the interaction of the oncogenic fusion protein MLLr with MEN1 has been shown to be essential. MEN1 binders inhibiting the MEN1 and KMT2A interaction have been shown to be effective against MLLr AML and B-ALL in experimental models and clinical studies. We hypothesized that in addition to the MEN1-KMT2A interaction, alternative mechanisms might be instrumental in the MEN1 dependency of leukemia. We first mined and analyzed data from publicly available gene expression databases, finding that the dependency of B-ALL cell lines on MEN1 did not correlate with the presence of MLLr. Using shRNA-mediated knockdown, we found that all tested B-ALL cell lines were sensitive to MEN1 depletion, independent of the underlying driver mutations. Most multiple myeloma cell lines that did not harbor MLLr were also sensitive to the genetic depletion of MEN1. We conclude that the oncogenic role of MEN1 is not limited to the interaction with KMT2A. Our results suggest that targeted degradation of MEN1 or the development of binders that induce global changes in the MEN1 protein structure may be more efficient than the inhibition of individual MEN1 protein interactions.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P<0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p < 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

T-Bet Overexpression Increases 4-1BB-Costimulated CAR T Cell Potency

Data from our lab and others has demonstrated that CAR T cells form a mixed Th1/Th2 population which, in contrast to canonical T cell differentiation, produce both interferon gamma (IFN-γ) and IL-4. Whereas a growing body of evidence has highlighted the importance of IFN-γ in the CAR T cell response, IL-4 can have immune-suppressive functions. IFN-γ from CD4+ CAR T cells (CAR4s), has been shown to promote cytotoxic effector functions of CD8+ CAR T cells (CAR8s), as well as bolster activation of endogenous immune cells including CD8 T cells and NK cells (Boulch et al Sci Immunol. 2021). Given the importance of IFN-γ to effective anti-tumor immune responses, we generated murine CAR T cells that target CD19 with a CD28 costimulatory domain (1928z) and compared them to CAR T cells that overexpress T-bet (T-bet-1928z) in an effort to skew CAR T cells away from IL-4 production and towards IFN-γ production. Upon stimulation of CAR T cells with the murine B-cell acute lymphoblastic leukemia (B-ALL) cell line E2A-PBX, fewer T-bet-1928z CAR4s produced IL-4 compared to 1928z CAR4s. This suggests T-bet overexpression is a viable strategy to reduce Th2-like programming of effector functions in CAR4s. To determine the impact of T-bet overexpression on early CAR T cell efficacy in vivo, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at peak expansion of CAR T cells as well as post-contraction. Both CARs eliminated CD19+ cells from the bone marrow and mediated long-term survival. T-bet-1928z CAR4s demonstrated increased expansion at Day 4 compared to 1928z CAR4s (p=0.0412), although there was no significant difference in CAR8 numbers at this timepoint. At Day 11 post-CAR, we observed an increase in the number of T-bet-1928z CAR T cells present in the bone marrow (p<0.0001), observed in both CAR4s (p<0.0001) and CAR8s (p=0.0074). While levels of memory-associated transcription factors TCF1 and FOXO1 were the same between CARs, fewer T-bet-1928z CAR T cells expressed IL7Rα (p=0.0002) and eomesodermin (p<0.0001), which are associated with the ability of T cells to self-renew and persist in the bone marrow. To determine the impact of T-bet on the persistence and renewal capacity of 1928z CAR T cells, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing RAG1-knockout recipients to eliminate endogenous T cell responses to E2A-PBX. After initial leukemia clearance, we rechallenged these mice with E2A-PBX and analyzed the bone marrow after 13 days. Both CARs were able to clear the leukemia rechallenge. While CAR4s were present in the bone marrow at similar numbers, the number of CAR8s was significantly higher among mice treated with T-bet-1928z CAR T cells (p=0.0317). Our results suggest that while T-bet may enhance in vivo expansion of CAR T cells, it provides no long-term benefit or deficit to the CAR T cell response. 4-1BB co-stimulated CAR T cells are less potent than CD28-costimulated CAR T cells and have reduced effector functions. We therefore hypothesized that T-bet overexpression would improve the efficacy of 4-1BB-costimulated CAR T cells by boosting effector functions and potency. To test this, we transduced T cells with a CAR targeting CD19 that contains a 4-1BB costimulatory domain (19BBz), or a 19BBz CAR and T-bet (19BBz-T-bet). We adoptively transferred 19BBz or 19BBz-T-bet CAR T cells into leukemia-bearing C57Bl6/J recipients. Despite no difference in CAR T cell numbers in the bone marrow, we observed fewer leukemia cells in mice treated with 19BBz-T-bet CAR T cells (p=0.0318) at Day 4 post-CAR. Treatment with 19BBz-T-bet CAR T cells also significantly improved survival of leukemia-bearing mice compared to 19BBz CAR T cells (p=0.0494). Our data demonstrate T-bet overexpression improves CD28-costimulated CAR T cell expansion but does little to augment long-term responses and diminishes features associated with capacity for self-renewal. In contrast, T-bet overexpression boosts the potency of 4-1BB-costimulated CAR T cells without impairing persistence, resulting in improved overall efficacy of 4-1BB-costimulated CAR T cell therapies.

Synthesis, anti-leukemia activity, and molecular docking of novel 3,16-androstenedione derivatives

In this study, we synthesized androsta-4,14-diene-3,16-dione, 12β-hydroxyandrosta-4,14-diene-3,16-dione, and other 3,16-androstenedione derivatives from commercially available dehydroepiandrosterone as a starting material in 9-13 steps with high yields. The bioactivity of the obtained compounds was evaluated. Compounds 14a and 23a were shown to have high antitumor activity against acute lymphoblastic leukemia cell lines Nalm-6 and BALL-1, respectively. Network pharmacology analysis showed that the anti-leukemia activity of compounds 14a and 23a might be related to the JAK2, ABL1 protein, and PI3K/Akt signaling pathways. The molecular docking of compounds 14a and 23a identified possible active sites, with the lowest docking scores for PTGS2 and MAPK14, respectively. In addition, the absorption, distribution, metabolism, and excretion prediction results revealed the drug-likeness of the two compounds. Therefore, compounds 14a and 23a should be considered anti-leukemia candidates in future studies.

CD19 chimeric antigen receptor T cell therapy in leukemia xenograft mouse: Anti-leukemic efficacy, kinetics, and 4-week single-dose toxicity

CD19 Chimeric antigen receptor T (CAR-T) cell therapy has shown a promising response rate for relapsed/refractory B-cell malignancies. However, serious side effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome arose in early case reports. Though several preclinical and clinical studies of CAR-T cell therapy have been reported, there is a lack of toxicological assessments. This study was carried out as a preclinical assessment of CD19 CAR-T cell therapy, including the anti-leukemic efficacy, kinetics in peripheral blood, and 4-week single-dose toxicity evaluation in leukemia xenograft mice. Leukemia xenograft mice model was established by injecting 1.0 × 105 cells/mouse of luciferase-labeled human B cell acute lymphoblastic leukemia (B-ALL) cell line via the tail vein, and after 3 days, 2.0 or 4.0 × 106 cells/mouse of CD19 CAR-T cells were injected intravenously. CD19 CAR-T cells showed significant anti-leukemic efficacy, showing inhibition of tumor progression in the bioluminescence-based in-vivo imaging system. In the kinetics study using qPCR, CAR-T cells peaked in peripheral blood on day 60 in males and day 30 in females. In a 4-week single-dose toxicity study, CD19 CAR-T cell injected groups showed no mortality and toxicological signs, or changes in body weight, food/water consumption, hematology, clinical chemistry, organ weights, and histopathology compared to control groups. These results suggested that 4.0 × 106 cells/mouse of CD19 CAR-T cells were effective in B-ALL xenograft mice without serious side effects, so the no-observed adverse effect level (NOAEL) was estimated to be higher than 4.0 × 106 cells/mouse, under the condition examined in the current study.

Bcl-2 family inhibitors sensitize human cancer models to therapy

BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.

Ruxolitinib inhibits the proliferation and induces the apoptosis of MLL-r ALL cells through inactivating JAK/STAT signaling pathway.

The childhood patients with mixed-lineage leukemia rearrangement (MLL-r) gene have worse outcome than non-MLL, and thus often treated with high-risk chemotherapy regimens, so targeted therapy is important for this type of leukemia. This purpose of study was to explore the effects of ruxolitinib on the proliferation, apoptosis, and cell cycle of Nalm-6 cells. In this study, human acute lymphoblastic leukemia (ALL) cell line Nalm-6 was used as the research object. By constructing an MLL overexpression vector to transfect Nalm-6 cells, exogenous JAK2/STAT3 signal pathway inhibitor ruxolitinib was applied to observe the proliferation, apoptosis, and cell cycle changes of the transfected Nalm-6 cells. Western blot was performed to determine the proteins (MLL-BP, JAK, STAT) involved in the mechanism of action of MLL-r leukemia. CCK8 assay and flow cytometry (FCM) were used for testing the proliferation and apoptosis among MLL-BP transfected Nalm-6 cells. Firstly, we determine the IC50 of ruxolitinib on Nalm-6 cells. Secondly, FCM and CCK8 showed that ruxolitinib dosedependentlyinhibits proliferation of Nalm-6 cells by blocking the cell cycle at G0/G1 phase. In addition, FCM showed that ruxolitinib promoted the apoptosis of MLL-BP transfected Nalm-6 cells. Mechanistically, ruxolitinib inactivated JAK/STAT signaling pathway in MLL-BP transfected Nalm-6 cells, mediating ruxolitinib's inhibition of cell proliferation, and inducing apoptosis. Finally, ruxolitinib significantly inhibited the proliferation of MLL-r ALL cells and promoted their apoptosis. These data provide compelling evidence that ruxolitinib is a promising agent against MLL-r leukemia cell line. However, it needs going through multiple more steps to confirm before it can be an option in clinical practice.

Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-β Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia

To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-β on T cell acute lymphoblastic leukemia (T-ALL) cell lines. Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. Different concentrations of FA-2-b-β significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-β inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-β reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/β-catenin inhibitor (ICG001), the process was further intensified. Agaricus Blazei Extract FA-2-b-β inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

Development and evaluation of novel artemisinin-isatin hybrids with potential anti-leukemic cytotoxicity.

Twenty-one novel ester tethered artemisinin-isatin hybrids were designed, synthesized and screened against human myeloid leukemia cell lines (K562 and K562/ADR), human acute lymphoblastic leukemia cell line (CCRF-CEM) as well as normal human peripheral blood mononuclear cells (PBMCs) for their cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The structure-activity relationships (SARs) were also discussed to facilitate further rational design of more effective candidates. The preliminary results showed that most of the ester tethered artemisinin-isatin hybrids (IC50: 0.32-29.35 µM) exhibited promising activity against CCRF-CEM cells, and some of them (IC50: 1.23-49.84 µM) were also active against K562 and K562/ADR human myeloid leukemia cell lines. Among them, hybrid 7d (IC50: 0.32, 2.67 and 1.23 µM) not only possessed profound activity against the three tested leukemia cell lines and excellent safety and selectivity profiles, but also showed promising pharmacokinetic properties. Accordingly, hybrid 7d could be considered as a potential lead molecule for the development of novel anti-leukemic agents with minimal untoward events to normal human cells.

Characterization of a novel MEF2D-BCL9 fusion-positive acute lymphoblastic leukemia cell line.

Not available.

Abstract 913: Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies

Abstract Despite the approval of three autologous CAR-T cell products, B-cell malignancies still represent an unmet need as several of the relapsed/refractory patients are ineligible for autologous CART cell therapies. Invariant natural killer T (iNKT) cells are a unique subset of immune cells that display properties of both T cells and natural killer cells and bridge the innate and adaptive immune responses upon antigen recognition. Unlike conventional β T cells, iNKT cells express an invariant T cell receptor (TCR) that recognize glycolipids presented in the context of the monomorphic, MHC-class I related molecule, CD1d. iNKT cells are ideal cell types for allogeneic, off the shelf approach as they can target cancers without the risk of graft-versus-host disease (GvHD), circumventing the need to delete or knock out the endogenous TCR. Here, we report the engineering of ‘off-the-shelf’ iNKT cells directed against CD19 and their robust in vitro and in vivo functional profile. Briefly, peripheral blood derived iNKT cells were isolated from healthy donors and were engineered to express a CD19 CAR using a 3rd generation lentiviral vector. To demonstrate the CAR-dependent and independent anti-tumor activity, CAR19-iNKT cells were compared in vitro against non-transduced (NT) iNKT cells in cytotoxicity assays and intracellular cytokine staining assays. Allogeneic CAR19-iNKT cells upregulated activation markers, secreted cytokines, and lysed cancer cell lines and primary tumor cells from chronic lymphocytic leukemia and marginal zone lymphoma patients in an antigen-specific manner. Excellent post thaw recovery and viability was noted in cryopreserved CAR19-iNKT cells. Finally, the anti-tumor activity of cryopreserved CAR19-iNKT cells were evaluated in an established aggressive NSG mice model of SEM-luc, a B cell lymphoblastic leukemia cell line expressing luciferase. Robust antitumor activity by CAR19-iNKT cells was observed without any obvious adverse effects compared to controls (untreated and NT iNKT cells). The persistence of CAR19-iNKT cells in this model is currently being evaluated. These data demonstrate a unique, off the shelf CAR19-iNKT cell platform for targeting hematological malignancies that combines the specificity of CD19 targeting CAR with the innate features of iNKT cells. The data indicates that iNKT cells are a potent cell type for generating allogeneic off the shelf products for treating a broad spectrum of hematological malignancies and solid tumors by targeting different receptors on the respective tumors. Citation Format: Kanagaraju Ponnusamy, Janani Kanagaraju, Yingxin Wang, Bryan Lye, Hongwei Ren, Sandhya Buchanan, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies [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 913.

Deep thermal profiling for detection of functional proteoform groups.

The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.

FBXW7 tumor suppressor regulation by dualspecificity tyrosine-regulated kinase 2

FBXW7 is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a main tumor suppressor due to its ability to control proteasome-mediated degradation of several oncoproteins such as c-Jun, c-Myc, Cyclin E1, mTOR, and Notch1-IC. FBXW7 inactivation in human cancers results from a somatic mutation or downregulation of its protein levels. This work describes a novel regulatory mechanism for FBXW7 dependent on the serine/threonine protein kinase DYRK2. We show that DYRK2 interacts with and phosphorylates FBXW7 resulting in its proteasome-mediated degradation. DYRK2-dependent FBXW7 destabilization is independent of its ubiquitin ligase activity. The functional analysis demonstrates the existence of DYRK2-dependent regulatory mechanisms for key FBXW7 substrates. Finally, we provide evidence indicating that DYRK2-dependent regulation of FBXW7 protein accumulation contributes to cytotoxic effects in response to chemotherapy agents such as Doxorubicin or Paclitaxel in colorectal cancer cell lines and to BET inhibitors in T-cell acute lymphoblastic leukemia cell lines. Altogether, this work reveals a new regulatory axis, DYRK2/FBXW7, which provides an understanding of the role of these two proteins in tumor progression and DNA damage responses.

Evaluation the Effect of Chemotherapy Drugs and Thiosemicarbazone Complexes on the Expression of URHC and CASC15 LncRNAs in Acute Lymphoblastic Leukemia Cell Line

Evaluation the Effect of Chemotherapy Drugs and Thiosemicarbazone Complexes on the Expression of URHC and CASC15 LncRNAs in Acute Lymphoblastic Leukemia Cell Line

Use of Novel Triphenyltin (IV) Dithiocarbamate Compounds in Assessing the Cytotoxicity and Mode of Cell Death of Acute Lymphoblastic Leukemia Cell Lines, CCRF CEM (CCL-119)

Chemotherapy and radiotherapy are often used to treat Acute Lymphoblastic Leukemia (ALL). However, the resistance problem of treatment using the current drug in ALL patients has decreased the effectiveness of such therapy. Therefore, a study needs to be conducted to identify the potential of new anticancer agents. Organotin (IV) dithiocarbamate, an organometallic compound, causes toxic effects on cancer cells in vitro and in vivo. This study examines the cytotoxic effects and mode of cell death of novel organotin (IV) dithiocarbamate compounds of triphenyltin (IV) diisopropyldithiocarbamate (compound 1), triphenyltin (IV) diallyldithiocarbamate (compound 2) and triphenyltin (IV) diethyldithiocarbamate (compound 3) on the acute lymphoblastic leukemia cell line, CCRF CEM (CCL-119). An MTT assay was used for the determination of the cytotoxicity of the compounds by treating cells with compounds 1-3 at the highest concentration of 10 µm for 24 h. The induction of the mode of cell death for compounds 1, 2, and 3 were identified using Annexin V-FITC/PI assay. From the results, all triphenyltin (IV) dithiocarbamate compounds have substantial cytotoxic effects with IC50 values between 0.18-0.20 μm. The chemicals induced apoptosis cell death in CCL-119 cells, based on the determination of cell death mode. However, statistical analysis showed that only apoptotic cell death for treatment with compounds 1-2 is significant compared to negative controls. In conclusion, all these compounds have a high cytotoxicity effect and are able to cause cell death via apoptosis in CCL-119 cells.

LncRNA15691 promotes T-ALL infiltration by upregulating CCR9 via increased MATR3 stability.

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration.

CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses

BackgroundTo date, T cells redirected with CD19-specific chimeric antigen receptors (CAR) have gained impressive success in B-cell malignancies. However, treatment failures are common and the occurrence of severe toxicities, such...