scholarly journals Adriamycin/Nucleophosmin Binding Protein-Conjugated Nanoparticle (ADR-PMs-NPMBP) Enhances Anti-Leukemia Activities of Adriamycin in Acute Lymphoblastic Leukemia Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-16
Author(s):  
Yingyu Chen ◽  
Donghui Gan ◽  
Liping Luo ◽  
Zhengjun Wu ◽  
Yuwen Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Drug Delivery System ◽  
Cell Lines ◽  
Delivery System ◽  
Binding Protein ◽  
Lymphoblastic Leukemia ◽  
Model Systems

Acute lymphoblastic leukemia (ALL) is an aggressive malignancy. Adults with ALL have more than 50% relapse rates and 20-40% overall survival. We previously validated that overexpression of NPM may be involved in the multidrug resistance development and might be important indicator for prognosis evaluation in ALL. In this study, we engineered and synthesized recombinant NPM binding protein (NPMBP). NPMBP and adriamycin (ADR) were bound to polymeric nanoparticle-based drug delivery system and named ADR-PMs-NPMBP (China Patent Application 202010302395.6, 202010739575.0). In this study, we aimed to evaluate thein vitroandin vivoanti-leukemia activity of ADR-PMs-NPMBP in resistant ALL cells. The underlying mechanisms were further addressed. In vitrostudy data demonstrated that ADR-PMs-NPMBP significantly enhanced chemosensitivity of ALL cells to ADR. The IC50 values of ADR-PMs-NPMBP in 4 ALL cell lines and primary ALL cells from 6 patients were between 1.6- and 7.0-fold lower than those of ADR. Of note, all the resistant ALL cell lines and the primary ALL cells from the relapsed patients, although variable in degree, were sensitive to ADR-PMs-NPMBP. The ratio of apoptosis cells in resistant ALL cells was more than 2-fold as compared ADR-PMs-NPMBP with single ADR regimen. RNA-seq analysis showed that there were 5599 and 233 differentially expressed genes in ADR-PMs-NPMBP versus control and ADR-PMs-NPMBP versus ADR comparison groups, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that 21 pathways play significant roles in ADR-PMs-NPMBP-mediated anti-leukemia activities in ALL. Of note, p53 protein with gene symbol TP53 was interestingly identified to overlap in the 5 pathways as following: Epstein-Barr virus infection, cell cycle, apoptosis, human T-cell leukemia virus 1 infection and p53 signaling pathway. Further validation experiments showed that ADR-PMs-NPMBP-induced effects on cell viability were due to the multiple roles of the activated p53 in apoptosis induction and cell cycle arrest. In vivostudy showed that ADR-PMs-NPMBP regimen was well tolerated by both resistant ALL xenograft BALB/C-nude model and ALL cell transplanted NOD-Prkdcem26Il2rgem26Nju (NCG) model. No systemic toxicity occurrence was observed after the recipients were intraperitoneally administered with ADR-PMs-NPMBP at 25 mg/kg/day for 3 days. The body weights of the animals increased with the time in both tumor model systems. Of note, whole-body bioluminescence imaging showed that less tumor cell burden was observed in ADR-PMs-NPMBP-treated recipients as compared to the animals administered with ADR. The tumor inhibition rate achieved as high as 79.2% in ADR-PMs-NPMBP group when finished the following-up. Moreover, studies on NCG model showed that ADR-PMs-NPMBP conditioning sensitized the antileukemia response of the resistant ALL xenograft model to ADR. Wright-Giemsa staining results demonstrated that more mature myeloid cells and lymphocytes were presented in the bone marrow samples from ADR-PMs-NPMBP-conditioned mice. In contrast, the increased immature blast cells were observed in the bone marrow from vehicle control mice and ADR-treated mice. Notably, the regimen of ADR-PMs-NPMBP diminished or blocked simultaneous metastases to multiple organs including liver and spleen, and significantly prolonged mouse survival. In conclusion, our study provides the first evidence that ADR-PMs-NPMBP nanoparticle may exert apoptotic inductionviathe activation of p53 pathway and markedly improve the antileukemia effect of ADR in resistant ALL cellsin vitroandin vivo. Our data indicate that the novel drug delivery system may be valuable to develop as a new strategy for the treatment of ALL, particularly for the resistant ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Doxorubicin/Nucleophosmin Binding Protein-Conjugated Nanoparticle Enhances Anti-leukemia Activity in Acute Lymphoblastic Leukemia Cells in vitro and in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Donghui Gan ◽  
Yuwen Chen ◽  
Zhengjun Wu ◽  
Liping Luo ◽  
Shimuye Kalayu Yirga ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Acute Lymphoblastic Leukemia ◽  
Drug Delivery System ◽  
Delivery System ◽  
Binding Protein ◽  
Lymphoblastic Leukemia ◽  
Multidrug Resistant ◽  
Apoptosis Induction

Acute lymphoblastic leukemia (ALL) is an aggressive malignancy. Adults with ALL have more than 50% relapse rates. We have previously validated that overexpression of nucleophosmin (NPM) is involved in the multidrug resistance (MDR) development during ALL; and a synthetically engineered recombinant NPM binding protein (NPMBP) has been developed in our group; NPMBP and doxorubicin (DOX) can be conjugated in a nanoparticle-based drug delivery system named DOX-PMs-NPMBP to counteract MDR during ALL. Here, we evaluated the antileukemia potential of DOX-PMs-NPMBP in resistant ALL cells. This study demonstrates that DOX-PMs-NPMBP significantly enhances chemosensitivity to DOX in ALL cells. Despite at variable concentrations, both resistant and primary ALL cells from relapsed patients were sensitive to DOX-PMs-NPMBP. In detail, the half maximal inhibitory concentration (IC50) values of DOX-PMs-NPMBP were between 1.6- and 7.0-fold lower than those of DOX in cell lines and primary ALL cells, respectively; and apoptotic cells ratio was over 2-fold higher in DOX-PMs-NPMBP than DOX. Mechanistically, p53-driven apoptosis induction and cell cycle arrest played essential role in DOX-PMs-NPMBP-induced anti-leukemia effects. Moreover, DOX-PMs-NPMBP significantly inhibited tumor growth and prolonged mouse survival of ALL xenograft models; and no systemic toxicity occurrence was observed after treatment during follow-up. In conclusion, these data indicate that DOX-PMs-NPMBP may significantly exert growth inhibition and apoptosis induction, and markedly improve DOX antileukemia activity in resistant ALL cells. This novel drug delivery system may be valuable to develop as a new therapeutic strategy against multidrug resistant ALL.

scholarly journals CALLA-positive myeloma: an aggressive subtype with poor survival

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 229-232
Author(s):  
BG Durie ◽  
TM Grogan
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Median Survival ◽  
Lymphoblastic Leukemia ◽  
Myeloma Cell ◽  
Negative Group ◽  
Poor Survival ◽  
Aggressive Subtype ◽  
Leukemia Antigen

Detailed immunotyping was carried out on 21 direct myeloma bone marrow aspirates and eight human myeloma cell lines. Four previously untreated common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma patients were identified and six of eight cell lines (75%) were also positive. CALLA positivity, as part of an immature B phenotype, was found to correlate with very aggressive clinical disease: median survival six months v 56 months for the CALLA-negative group.

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3699-3699
Author(s):  
Na Yuan ◽  
Lin Song ◽  
Suping Zhang ◽  
Weiwei Lin ◽  
Yan Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Western Blotting ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Bafilomycin A1 ◽  
Low Concentration

Abstract The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

The Wee1 Inhibitor, MK-1775, Sensitizes Leukemic Cells to Different Antineoplastic Drugs Interfering with DNA Damage Response Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1276-1276 ◽  
Author(s):  
Andrea Ghelli Luserna Di Rora ◽  
Ilaria Iacobucci ◽  
Neil Beeharry ◽  
Simona Soverini ◽  
Cristina Papayannidis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Additive Effect ◽  
Proliferation Index ◽  
Induction Of Apoptosis

Abstract Due to inadequate treatments, the survival rate of adult Acute Lymphoblastic Leukemia (ALL) patients with the exclusion of patients with particular genetic alterations, like the Philadelphia positive patients, is still very low. Moreover even the rate of patient that responds to specific treatment develops relapses during their life. Thus there is a need to improve the efficacy of conventional therapy and to discover novel specific targets. In eukaryotic cells Wee1, ATR/Chk1 and ATM/Chk2 are three pathways involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the inhibitory phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) respectively. This study evaluates the effectiveness of MK-1775, a selective Wee1 inhibitor, as a monotherapy and as chemosensitizer agent for the treatment of B-/T-Acute Lymphoblastic Leukemia. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were tested in this study. MK-1775 alone strongly reduced the cell viability in a dose and time-dependent manner in all the cell lines treated. The anti-proliferative activity of MK-1775 was accompanied by an increase in apoptotic cells (AnnexinV/Pi staining) and by DNA damage markers (gH2AX and Parp-1 cleavage). Moreover the inhibition of Wee1 disrupted the cell cycle profile by arresting the cells in late S and in G2/M phase. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. Moreover the immunofluorescence staining for the DNA damage marker gH2AX and the mitotic marker phosphor-Histone H3 showed that co-treatment with MK-1775 and PF-0477736 induced cell death by mitotic catastrophe. We undertook further studies to understand the immediate clinical potential of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, and a particular isomer of this compound).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bosutinib and Bos-isomer (Bos-I). Both the isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability. Interestedly the cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bosutinib authentic, interfered with the Chk1/Chk2 and Wee1 pathway. This supported our previous studies showing that Bosutinib and its isomer possess off-target effects against both Wee1 and Chk1 kinases and thus maybe used as a chemosensitizer (Beeharry et al. Cell Cycle 2014). The results of this study in our opinion identify the Wee1 kinase as a promising target for the treatment of ALL not only as a monotherapy but also as chemosensitizer agent to increase the cytotoxicity of different kind of drugs already used in clinical trials. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy.

The Inhibition of Checkpoint Kinase 1 As a Promising Strategy to Increase the Effectiveness of Different Treatments in Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2478-2478
Author(s):  
Andrea Ghelli Luserna Di Rora ◽  
Ilaria Iacobucci ◽  
Enrica Imbrogno ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Dna Damages ◽  
Checkpoint Kinase

Abstract Nowadays the effectiveness of the treatments for adult Acute Lymphoblastic Leukemia (ALL) patients is still inadequate and frequently many patients after years of response to treatments develop relapses. Thus there is a need to find novel targets for specific therapies and to maximize the effect of the actual treatments. Recently different Checkpoint Kinase (Chk)1/Chk2 inhibitors has been assessed for the treatment of different type of cancers but only few studies have been performed on hematological diseases. We evaluated the effectiveness of the Chk1 inhibitor, LY2606368, as single agent and in combination with tyrosine kinase inhibitors (imatinib and dasatinib) or with the purine nucleoside antimetabolite clofarabine in B-/T- acute lymphoblastic leukemia (ALL) cell lines and in primary blasts. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were incubated with increasing concentrations of drug (1-100 nM) for 24 and 48 hours and the reduction of the cell viability was evaluated using WST-1 reagent. LY2606368 deeply reduced the cell viability in a dose and time dependent manner in all the cell lines, with the BV-173 (6.33 nM IC50 24hrs) and RPMI-8402 (8.07 nM IC50 24hrs) being the most sensitive while SUP-B15 (61.4 nM IC50 24hrs) and REH (96.7 nM IC50 24hrs) being the less sensitive cell lines. Moreover the sensitivity to the compound was no correlated with the different sub-type of ALL or with the mutational status of p53, which is a marker of the functionality of the G1/S checkpoint. The cytotoxic activity was confirmed by the significant increment of apoptosis cells (Annexin V/Propidium Iodide), by the increment of gH2AX foci and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). To understand the relationship between the activation of apoptosis and the effect on cell cycle and to identify hypothetical mechanisms of death, different cell cycle analyses were performed (Propidium Iodide staining). The inhibition of Chk1, deeply changed the cell cycle profile. Indeed in all the cell lines the percentage of cells in S phase and in G2/M phase were reduced by the treatment while the numbers of cells in sub-G1 and G1 phase were increased. The hypothetical function of LY2606368 as a chemosensitizer agent was evaluated combining the compound with different drugs normally used in clinical trials. For each drugs the combination strongly reduced the cell viability when compared to the cytotoxic effect of the single drugs. Moreover the combination showed an additive efficacy in term of induction of DNA damages as showed by the increase number of gH2AX foci and the activation of pChk1 (ser 317). The results found on the cell lines were confirmed also using primary leukemic blast isolated from adult Philadelphia-positive ALL patients. Indeed LY2606368 as single agent or in combination with the Tki, imatinib, was able to deeply reduce the cell viability and to induce DNA damages (gH2AX foci). In conclusion LY2606368 showed a strong cytotoxic activity on B-/T-All cell lines and primary blasts as single agent and in combination with other drugs. In our opinion this data are the basis for a future clinical evaluation of this compound in the treatment of leukemia. Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:ROCHE: Consultancy; Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

scholarly journals Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells

2021 ◽  
Author(s):  
Andrea Ghelli Luserna Di Rorà ◽  
Martina Ghetti ◽  
Lorenzo Ledda ◽  
Anna Ferrari ◽  
Matteo Bocconcelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Cycle Checkpoint ◽  
M Cell ◽  
Dna Damages ◽  
Cycle Checkpoint ◽  
Dna Double Helix

AbstractDoxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. Graphical abstract • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell Analyses Identify Transcriptional Characterizations of Subsets Associated with Efficacy and Toxicity for CAR-T Immunotherapy in B-ALL Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4815-4815
Author(s):  
Mengyi Du ◽  
Heng Mei ◽  
Chenggong Li ◽  
Yinqiang Zhang ◽  
Lu Tang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Single Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Mrna Sequencing ◽  
Car T ◽  
Mononuclear Bone Marrow Cells ◽  
Marrow Cells

Abstract Background The development of mRNA sequencing has contributed greatly to the mechanism exploration in hematologic malignancies disease. With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize every subset of expression programs and functional states in a comprehensive and unbiased manner. Here, we present a systematic evaluation of engineered chimeric antigen receptor T (CAR-T) products and patient bone marrow profiles in terms of primary resistance and severe cytokine release syndrome (CRS) at the single-cell level. Methods Using single-cell mRNA sequencing in conjunction with flow cytometry (FCM), we performed characterization of CD19-targeted CAR-T and mononuclear bone marrow cells from 4 on-trial B acute lymphoblastic leukemia (B-ALL) patients (NCT02965092). Bioinformatics analysis was utilized to explore diversity between patients with different grades of response or CRS. Basing on marker genes, CAR-T products were divided into four groups, which were double-positive T (DPT), CD4 positive T (CD4), CD8 positive T (CD8), and double-negative T (DNT) cells. Meanwhile, both the mononuclear bone marrow cells before and after CAR-T infusion were grouped into six clusters, which were B-ALL, stem, progenitor, B, T, and myeloid cells. The expression and enrichment analyses results were calculated by R (version 3.6.3) and then verified in a 22-sample conventional transcription sequencing cohort of the same clinical trial. Patient efficacy was assessed by the national comprehension cancer network guidelines version 2.2020 for acute lymphoblastic leukemia, and CRS was graded by CTCAE 5.0. Results By FCM detection, the variances of CAR-T infusion products between patients with different clinical outcomes were limited, and nor did mononuclear bone marrow cells. The scRNA sequencing results showed that distinct CAR-T and bone marrow cell subsets indicated differentiated expression in proliferation, cytotoxicity, and intercellular signaling pathways. Expression differentiation variances in CAR-T infusion products were minor than in mononuclear bone marrow cells. CD8+ CAR-T products of complete response (CR) patients were still significantly enriched in pathways such as cell killing (p adjust=0.0012), antigen processing and presentation (p adjust=0.0027), and cell cycle (p adjust=0.0231), exhibiting greater immune function when compared with no response patients. Also, DPT CAR-T products of the non-CRS patients were meaningfully enriched in negative regulation of cytokine production pathway (p adjust=0.0127) when compared with CRS ones. In mononuclear bone marrow cells, B-ALL cells before CAR-T treatment of CR patients presented negatively in cell-cycle (p adjust=0.0019), leading to a low malignant cell proliferation level; and stem-progenitor cells after CAR-T treatment of CR patients showed a stronger ability of neutrophil activation (p adjust<0.0001). As with comparisons between CRS and non-CRS, B-ALL cells before infusion manifested a cell cycle arrest profile (p adjust<0.0006) in non-CRS patients, whereas the immune cells at the same time point were enriched in positive regulation of cell cycle process (p adjust=0.0002). Conclusions Through single-cell RNA-seq profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the cell cycle, immune phenotype, and metabolic profiles of subsets during CAR-T therapy, providing a mechanistic basis for ameliorating clinical outcomes and individualized management. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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