B-ALL Cells Research Articles

Reimagining petroleum refining.

Rigid polymer membranes separate crude oil components without distillation

ISSUE HIGHLIGHTS - July 2020.

ISSUE HIGHLIGHTS - July 2020.

Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model.

Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO2) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO2 in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO2 and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO2 in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.

Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-JUN N-terminal kinase inhibition

BackgroundThe Philadelphia chromosome (Ph), which leads to the creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) have shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph+ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph+ B-ALL.MethodsActivation of the JNK signaling pathway in human and mouse BCR-ABL+ B-ALL cells with or without dasatinib treatment was analyzed by Western blotting. JNK was inhibited either by RNA interference or chemical inhibitors, such as JNK-IN-8. The effect of JNK inhibition with or without BCR-ABL TKI dasatinib on BCR-ABL+ B-ALL cells was analyzed by the CellTiter-Glo® Luminescent Cell Viability Assay. The in vivo effects of JNK-IN-8 and dasatinib alone or in combination were tested using a BCR-ABL induced B-ALL mouse model.ResultsWe found that the c-JUN N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL+ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph+ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph+ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone.ConclusionsOur findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph+ B-ALL.

Non-signaling Chimeric Antigen Receptors Enhance Antigen-Directed Killing by γδ T Cells in Contrast to αβ T Cells

Chimeric antigen receptor (CAR)-modified T cells have demonstrated efficacy against B cell leukemias/lymphomas. However, redirecting CAR T cells to malignant T cells is more challenging due to product-specific cis- and trans-activation causing fratricide. Other challenges include the potential for product contamination and T cell aplasia. We expressed non-signaling CARs (NSCARs) in γδ T cells since donor-derived γδ T cells can be used to prevent product contamination, and NSCARs lack signaling/activation domains, but retain antigen-specific tumor cell-targeting capability. As a result, NSCAR targeting requires an alternative cytotoxic mechanism, which can be achieved through utilization of γδ T cells that possess major histocompatibility complex (MHC)-independent cytotoxicity. We designed two distinct NSCARs and demonstrated that they do not enhance tumor-killing by αβ T cells, as predicted. However, both CD5-NSCAR- and CD19-NSCAR-modified γδ T cells enhanced cytotoxicity against T and B cell acute lymphoblastic leukemia (T-ALL and B-ALL) cell lines, respectively. CD5-NSCAR expression in γδ T cells resulted in a 60% increase in cytotoxicity of CD5-expressing T-ALL cell lines. CD19-NSCAR-modified γδ T cells exhibited a 350% increase in cytotoxicity against a CD19-expressing B-ALL cell line compared to the cytotoxicity of naive cells. NSCARs may provide a mechanism to enhance antigen-directed anti-tumor cytotoxicity of γδ T cells through the introduction of a high-affinity interaction while avoiding self-activation.

Reprogramming leukemia cells into antigen presenting cells as a novel cancer vaccination immunotherapy

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in malignant transformation. Despite an arrest in B cell maturation, human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage. To test the therapeutic implications of reprogramming, we generated murine models of B-ALL capable of myeloid lineage reprogramming via ectopic expression of CEBPα and PU.1. Once reprogrammed, B-ALL cells acquired an APC phenotype and stimulated antigen-specific T cells. In vivo B-ALL reprogramming in immunodeficient mice led to a modest survival benefit, however, reprogramming in immunocompetent mice led to tumor eradication and protection from subsequent re-challenge, demonstrating successful generation of durable, systemic, and curative immunity. This therapeutic benefit is dependent on the presence of both CD4+ and CD8+ T cells, and characterized by an increased frequency of bone marrow-infiltrating T cells and oligoclonal T cell expansion. Moreover, local myeloid reprogramming of a primary tumor drove systemic immune activation and eradication of distant, non-reprogrammed lesions. Our data suggests that B-ALL cells reprogrammed to the myeloid lineage are potent APCs capable of presenting tumor-associated antigens, and in vivo reprogramming elicits robust, durable, tumor-eradicating, and systemic T cell-mediated immunity. Recent efforts have further identified sarcoma and carcinoma models amenable to myeloid-lineage reprogramming. Thus, reprogramming of malignant cells into APCs represents a novel immunotherapeutic strategy with potential clinical utility in the treatment of a broad array of human malignancies.

Promoting anti-tumor immunity via bispecific T cell engaging cytokine (biteokine) therapy

Abstract Bispecific T cell engager (BiTE) therapy bypasses the need for natural recognition of tumor cells by cytotoxic T cells, inducing potent anti-tumor immunity. Currently, the FDA approved BiTE, Blinatumomab (Blin), provides an alternative to standard chemotherapy in patients with relapsed/refractory or MRD+ Acute Lymphoblastic Leukemia (ALL) and is in clinical trials for other B cell malignancies. Blin targets malignant B cells using the antigen binding domain of an αCD19 antibody linked to the antigen binding domain of a T cell-activating αCD3 antibody. Despite progress with Blin clinically, complete response is 39–69%, and a significant proportion of patients are refractory or relapse. To improve this outcome, we have engineered a nanoparticle-based assembly and screening approach to capitalize on cytokine-enhancement of BiTE activity. We found that IL-12 significantly improves Blin-induced B-ALL cell lysis in vitro and, using nanoparticles capable of displaying ~142 antibodies/particle, we generated 28 unique formulations with varying densities and ratios of antibodies to target CD19, activate CD3, and localize IL-12 in a non-neutralizing fashion. We screened these particles in parallel using a co-culture assay for target cell lysis and T cell expansion by flow cytometry. Through regression modeling of these data, we can select-for and optimize architectures that maximize target cell lysis and T cell proliferation. Our preliminary results indicate that optimally lytic architectures favor high αCD3 to αCD19 ratios and are improved linearly with increasing IL-12. This work seeks to innovate and enhance bispecific T cell engager therapy with a modular platform that can be used broadly in the treatment of cancer and autoimmunity.

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis.

BackgroundAcute lymphocytic leukemia (ALL) is a common blood cancer which induces high mortality in children. Bromodomains and extra-terminal (BET) protein inhibitors, such as JQ1 and ARV-825, are promising cancer therapeutic agents that can be used by targeting c-Myc. A recent work reported that JQ1 effectively attenuates ALL in vitro by suppressing cell proliferation and accelerating apoptosis. The purpose of this research was to probe into the potential mechanism of how JQ1 inhibits ALL cell proliferation in vitro.Material/MethodsCell viability of ALL cells were measured by CTG after treatment by JQ1. Cell cycle analysis was done by EdU and PI staining. Cell apoptosis was assessed by Annexin V/PI staining. Glycolysis was detected using Seahorse and LC-MS kits. The expression of glycolytic rate-limiting enzymes was assessed by RNA-seq, qRT-PCR, and Western blot.ResultsJQ1 suppressed cell proliferation by arresting the cell cycle and inducing the apoptosis of acute lymphocytic leukemia cells. JQ1 inhibited cell proliferation of B-ALL cells by restraining glycolysis. Conversely, the cell cycle block of B-ALL cells induced by JQ1 was partially abolished after pretreatment with 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis. Furthermore, JQ1 restrained the glycolysis of B-ALL cell lines by remarkably downregulating the rate-limiting enzymes of glycolysis, such as hexokinase 2, phosphofructokinase, and lactate dehydrogenase A. Moreover, the cell cycle arrest was reversed in B-ALL cells with overexpressed c-Myc treated by JQ1, which is involved in the enhancement of glycolysis.ConclusionsThe BET inhibitor JQ1 suppresses the proliferation of ALL by inhibiting c-Myc-mediated glycolysis, thus providing a new strategy for the treatment of ALL.

17-DMAG disrupted the autophagy flux leading to the apoptosis of acute lymphoblastic leukemia cells by inducing heat shock cognate protein 70

AimsB-lineage acute lymphoblastic leukemia (B-ALL) is most common in children. We had reported heat shock protein 90 (Hsp90) over-expressed in high risk B-ALL children. 17-DMAG is a water soluble Hsp90 inhibitor, which was proved to be effective for advanced solid tumors and hematological malignancy. However, there is little research on its application in newly diagnosed B-ALL. And the detailed mechanism is seldom discussed. Main methodsPrimary blast cells from 24 newly diagnosed B-ALL pediatric patients and two B-ALL cell lines were used in this study. Cell viability was measured by MTS assay. Apoptosis was evaluated by flow cytometry after annexin V-PI double staining. Protein expression was detected by immunoblotting analysis and immunofluorescence imaging. Cyto-ID autophagy detection assay was performed to show the autophagosomes and LysoTracker labeling to show the lysosomes. Gene knockdown was performed by RNA interference, and mRNA expression was measured by RT-qPCR. Key findingsWe showed 17-DMAG induced apoptosis in newly diagnosed B-ALL blasts and cell lines effectively. 17-DMAG induced heat shock cognate protein 70 (Hsc70) expression significantly. High expressed Hsc70 inhibited cathepsin D post-transcriptionally to impede the autophagic flux, which lead to the cell death. SignificanceOur work added new information towards understanding the molecular pharmacology of 17-DMAG, and suggested the newly diagnosed B-ALL pediatric patients might be benefited from 17-DMAG. Furthermore, we proved Hsc70 participated in the mechanism of cell death 17-DMAG leading in B-ALL.

Influence of Casein kinase II inhibitor CX-4945 on BCL6-mediated apoptotic signaling in B-ALL in vitro and in vivo

BackgroundCasein kinase II (CK2) is involved in multiple tumor-relevant signaling pathways affecting proliferation and apoptosis. CK2 is frequently upregulated in acute B-lymphoblastic leukemia (B-ALL) and can be targeted by the ATP-competitive CK2 inhibitor CX-4945. While reduced proliferation of tumor entities including B-ALL after CX-4945 incubation has been shown in vitro and in vivo, the detailed way of action is unknown. Here, we investigated the influence on the PI3K/AKT and apoptosis cascades in vivo and in vitro for further clarification.MethodsA B-ALL xenograft model in NSG mice was used to perform in vivo longitudinal bioluminescence imaging during six day CX-4945 treatment. CX-4945 serum levels were determined at various time points. Flow cytometry of bone marrow and spleen cells was performed to analyze CX-4945-induced effects on tumor cell proliferation and distribution in B-ALL engrafted mice. ALL cells were enriched and characterized by targeted RNA sequencing. In vitro, B-ALL cell lines SEM, RS4;11 and NALM-6 were incubated with CX-4945 and gene expression of apoptosis regulators BCL6 and BACH2 was determined.ResultsIn B-ALL-engrafted mice, overall tumor cell proliferation and distribution was not significantly influenced by CK2 inhibition. CX-4945 was detectable in serum during therapy and serum levels declined rapidly after cessation of CX-4945. While overall proliferation was not affected, early bone marrow and spleen blast frequencies seemed reduced after CK2 inhibition. Gene expression analyses revealed reduced expression of anti-apoptotic oncogene BCL6 in bone marrow blasts of CX-4945-treated animals. Further, BCL6 protein expression decreased in B-ALL cell lines exposed to CX-4945 in vitro. Surprisingly, levels of BCL6 opponent and tumor suppressor BACH2 also declined after prolonged incubation. Simultaneously, increased phosphorylation of direct CK2 target and tumor initiator AKT was detected at respective time points, even in initially pAKT-negative cell line NALM-6.ConclusionsThe CK2 inhibitor CX-4945 has limited clinical effects in an in vivo B-ALL xenograft model when applied as a single drug over a six day period. However, gene expression in B-ALL cells was altered and suggested effects on apoptosis via downregulation of BCL6. Unexpectedly, the BCL6 opponent BACH2 was also reduced. Interactions and regulation loops have to be further evaluated.

Targeting Two Antigens Associated with B-ALL with CD19-CD123 Compound Car T Cell Therapy.

The recent FDA approval of the first CAR immunotherapy marks a watershed moment in the advancement toward a cure for cancer. CD19 CAR treatment for B cell acute lymphocytic leukemia has achieved unprecedented remission rates. However, despite success in treating previously relapsed and refractory patients, CD19 CAR faces similar challenges as traditional chemotherapy, in that malignancy can adapt and overcome treatment. The emergence of both antigen positive and negative blasts after CAR treatment represents a need to bolster current CAR approaches. Here, we report on the anti-tumor activity of a CAR T cell possessing 2 discrete scFv domains against the leukemic antigens CD19 and CD123. We determined that the resulting compound CAR (cCAR) T cell possesses consistent, potent, and directed cytotoxicity against each target antigen population both in vitro and in vivo. Our findings indicate that targeting CD19 and CD123 on B-ALL cells may be an effective strategy for augmenting the response against leukemic blasts and reducing rates of relapse.

Differential Depletion of Bone Marrow Resident B-ALL after Systemic Administration of Endosomal TLR Agonists.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. While frontline chemotherapy regimens are generally very effective, the prognosis for patients whose leukemia returns remains poor. The presence of measurable residual disease (MRD) in bone marrow at the completion of induction therapy is the strongest predictor of relapse, suggesting that strategies to eliminate the residual leukemic blasts from this niche could reduce the incidence of recurrence. We have previously reported that toll-like receptor (TLR) agonists achieve durable T cell-mediated protection in transplantable cell line-based models of B cell precursor leukemia (B-ALL). However, the successful application of TLR agonist therapy in an MRD setting would require the induction of anti-leukemic immune activity specifically in the bone marrow, a site of the chemotherapy-resistant leukemic blasts. In this study, we compare the organ-specific depletion of human and mouse primary B-ALL cells after systemic administration of endosomal TLR agonists. Despite comparable splenic responses, only the TLR9 agonist induced strong innate immune responses in the bone marrow and achieved a near-complete elimination of B-ALL cells. This pattern of response was associated with the most significantly prolonged disease-free survival. Overall, our findings identify innate immune activity in the bone marrow that is associated with durable TLR-induced protection against B-ALL outgrowth.

Targeting BCL-2 as a Therapeutic Strategy for Primary p210BCR-ABL1-positive B-ALL Cells.

Philadelphia-positive acute lymphoblastic leukemia (Ph+ B-ALL) is caused by the malignant transformation of lymphoid cells induced by BCR-ABL1 constitutive catalytic activity. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML) cells, inducing durable hematological, cytogenetic and molecular responses. However, in Ph+ B-ALL - as in CML progressing to blast crisis - TKIs fail to maintain disease remission. We, therefore, wanted to investigate if dual targeting of BCL-2 and BCR-ABL1 would be more effective in killing Ph+ B-ALL cells. p210-B-ALL CD34-positive cells were used to evaluate the BCR-ABL expression and pharmacological targeting of BCL-2, by venetoclax, alone or in combination with BCR-ABL1 inhibition. We demonstrated the cytotoxic effect of BCL-2 inhibition and that dual targeting of BCL-2 and BCR-ABL1 with venetoclax and nilotinib further increases this cytotoxicity. BCL-2 is a key survival factor for primary Ph+ B-ALL cells and its inhibition - alone or in combination with a BCR-ABL1 TKI - should be further investigated as a potential therapeutic strategy for these patients.

Antitumor effect of baicalin from the Scutellaria baicalensis radix extract in B-acute lymphoblastic leukemia with different chromosomal rearrangements

Acute B-lymphoblastic leukemia (B-ALL) is the most common hematologic malignancy in children. Many cases of B-ALL harbor chromosomal translocations which are often critical determinants of prognosis. Most of them represent altered transcription factors that impact gene transcription or enhance signaling. B-ALLs harboring the mixed-lineage leukemia 1 (MLL1) gene rearrangements represent aggressive, high-risk type of early childhood leukemias that are usually associated with a very poor prognosis. Therefore, there is an urgent need for novel therapeutic agents as well as new treatment strategies.The objective was to examine the vitro inhibitory effects of Scutellaria baicalensis root extract (SBE) in B-ALL cell lines with different chromosomal rearrangements and in leukemic blasts derived from patients’ bone marrow (BMCs).In this study we showed that baicalin which is the main component of the SBE possess antitumor activity against all leukemic cell lines especially those with MLL and PBX1 gene rearrangements. Baicalin inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase, and induced cell death through caspase 3/7 activation. Moreover, baicalin treatment inhibited the glycogen synthase kinase-3β (GSK‐3β) by suppressing its phosphorylation at Y216, and upregulated the downstream mediator of the cell cycle arrest - cyclin dependent kinase inhibitor p27Kip1. Bone marrow derived blasts from B-ALL patients also exhibited varied sensitivity towards baicalin with 72% patients sensitive to the SBE and baicalin treatment.Taken together, our findings provide new insights into the anti-cancer properties of baicalin by showing its diverse mode of action which might be related to the different genetic background.

Let's Talk About BiTEs and Other Drugs in the Real-Life Setting for B-Cell Acute Lymphoblastic Leukemia.

Background: Therapy for acute lymphoblastic leukemia (ALL) are currently initially efficient, but even if a high percentage of patients have an initial complete remission (CR), most of them relapse. Recent data shows that immunotherapy with either bispecific T-cell engagers (BiTEs) of chimeric antigen receptor (CAR) T cells can eliminate residual chemotherapy-resistant B-ALL cells.Objective: The objective of the manuscript is to present improvements in the clinical outcome for chemotherapy-resistant ALL in the real-life setting, by describing Romania's experience with bispecific antibodies for B-cell ALL.Methods: We present the role of novel therapies for relapsed B-cell ALL, including the drugs under investigation in phase I-III clinical trials, as a potential bridge to transplant. Blinatumomab is presented in a critical review, presenting both the advantages of this drug, as well as its limitations.Results: Bispecific antibodies are discussed, describing the clinical trials that resulted in its approval by the FDA and EMA. The real-life setting for relapsed B-cell ALL is described and we present the patients treated with blinatumomab in Romania.Conclusion: In the current manuscript, we present blinatumomab as a therapeutic alternative in the bridge-to-transplant setting for refractory or relapsed ALL, to gain a better understanding of the available therapies and evidence-based data for these patients in 2019.

BAX Gene Deletion Reduces the Sensitivity of BCR-ABL-Induced B-ALL Cells of Mice to Imatinib

To investigate the effect of BAX gene deletion on the sensitivity of BCR-ABL-induced B-ALL cells of mice to imatinib and the related mechanism. The target gene-knock out (BAX-/-) mice were used as bone marrow cell donors; the wild type bone marrow cells(B6BM) and BAX-/- bone marrow cells(B6BM-BAX-/-) of mice were transfected by using reverse transcription virus, then the BCR-ABL transfected B6BM cells and B6BM-BAX-/- cells were treated with imatinib at different concentration (0,0.5, 1.0 and 2.0 μmol/L) for 48 hours. The number of viable cells was detected by trypan blue, the flow cytometry was used to detect the cell apoptosis, the Western blot was used to detect the changes of BAX, Caspase expression. In BCR-ABL transfected bone marrow cells treated with imatinib, the numbers of viable cells of BAX deletion group was significantly higher than that of wild type groups with statristcal difference(P<0.05), and effect- and dose-dependency(r=-0.9533 for BAX deletion group, and r=-0.9812 for wild type group). The flow cytometry showed that the cell apoptosis in BAX deletion group signifincantly decreased, compared with wild type group(P<0.05). The Western blot showed that the expression of apoptotic protein Caspase 3 in BAX deletion group was significantly higher than that in wild type group(P<0.05). BAX deletion can reduce the sensitivity of BCR-ABL-induced B-ALL cells to imatinib.

BAX Gene Deletion Reduces the Sensitivity of BCR-ABL-Induced B-ALL Cells of Mice to Imatinib

To investigate the effect of BAX gene deletion on the sensitivity of BCR-ABL-induced B-ALL cells of mice to imatinib and the related mechanism. The target gene-knock out (BAX-/-) mice were used as bone marrow cell donors; the wild type bone marrow cells(B6BM) and BAX-/- bone marrow cells(B6BM-BAX-/-) of mice were transfected by using reverse transcription virus, then the BCR-ABL transfected B6BM cells and B6BM-BAX-/- cells were treated with imatinib at different concentration (0,0.5, 1.0 and 2.0 μmol/L) for 48 hours. The number of viable cells was detected by trypan blue, the flow cytometry was used to detect the cell apoptosis, the Western blot was used to detect the changes of BAX, Caspase expression. In BCR-ABL transfected bone marrow cells treated with imatinib, the numbers of viable cells of BAX deletion group was significantly higher than that of wild type groups with statristcal difference(P<0.05), and effect- and dose-dependency(r=-0.9533 for BAX deletion group, and r=-0.9812 for wild type group). The flow cytometry showed that the cell apoptosis in BAX deletion group signifincantly decreased, compared with wild type group(P<0.05). The Western blot showed that the expression of apoptotic protein Caspase 3 in BAX deletion group was significantly higher than that in wild type group(P<0.05). BAX deletion can reduce the sensitivity of BCR-ABL-induced B-ALL cells to imatinib.

Relative safety of various spermatogenic stem cell purification methods for application in spermatogenic stem cell transplantation

BackgroundSpermatogonial stem cell (SSC) transplantation technology as a promising option for male fertility preservation has received increasing attention, along with efficient SSC purification technology as a necessary technical support; however, the safety of such application in patients with tumors remains controversial.MethodsIn this study, we used a green fluorescent protein mouse xenograft model of B cell acute lymphocytic leukemia. We isolated and purified SSCs from the testicular tissue of model mice using density gradient centrifugation, immune cell magnetic bead separation, and flow cytometry. The purified SSCs were transplanted into convoluted seminiferous tubules of the nude mice and C57BL/6 male mice subjected to busulfan. The development and proliferation of SSCs in the recipient testis were periodically tested, along with whether B cell acute lymphocytic leukemia was induced following SSC implantation. The genetic characteristics of the offspring obtained from natural mating were also observed.ResultsIn testicular leukemia model mice, a large number of BALL cells infiltrated into the seminiferous tubule, spermatogenic cells, and sperm cells in the testis tissue decreased. After spermatogonial stem cell transplantation, the transplanted SSCs purified by immunomagnetic beads and flow cytometry methods colonized and proliferated extensively in the basement of the seminiferous tubules of mice; a large number of spermatogenic cells and sperm were found in recipient testicular tissue after 12 weeks of SSC transplantation. In leukemia detection in nude mice after transplantation in the three SSC purification groups, a large number of BALL cells could be detected in the blood of recipient mice 2–3 weeks after transplantation in the density gradient centrifugation group, but not in the blood of the flow cytometry sorting group and the immunomagnetic bead group after 16 weeks of observation.ConclusionsIn this study, we confirmed that immunomagnetic beads and flow cytometry methods of purifying SSCs from the testicular tissue of the testicular leukemia mouse model could be safely applied to the SSC transplantation technology without concomitant tumor implantation. The results thus provide a theoretical basis for the application of tumor SSC cryopreservation for fertility preservation in patients with tumors.

Paraoxonase 2 Enables Initiation of B-ALL By Subverting Metabolic Gatekeeper Functions

Background and significance: The B-lymphoid transcription factors IKZF1 and PAX5 are essential for early B-cell development but also function as metabolic gatekeepers by restricting glucose uptake across the cell membrane (Chan et al., Nature 2017; Xiao et al., Cell 2018). Paraoxonase 2 (PON2) is used as a diagnostic marker and included in a 15 gene diagnostic LDA panel (NCT02883049) for the identification of Ph+ and Ph-like ALL, a B-ALL subgroup with poor outcome and frequent deletion of IKZF1. Pon2 is a member of detoxifying enzymes that are located to the mitochondrial membrane, and hydrolyze lactone metabolites. While PON2 is typically not expressed in normal B-cell precursors, we studied the functional role of high expression levels of Pon2 in B-ALL. Results: Indeed, high Pon2 mRNA levels at diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003). While PON2 mRNA and protein levels are barely detectable in normal B-cell precursors we found &amp;gt;10-fold increased mRNA and protein levels in multiple B-ALL. Pon2-deficient mice have a number of subtle defects, including propensity to neurogenerative disease and atherosclerosis. However, deletion of Pon2 had profound effects in two models for B-ALL (BCR-ABL1- and NRASG12D). Compared to Pon2 wildtype, Pon2-/- B-ALL cells failed to form colonies in semisolid agar, were prone to cell cycle arrest in G0/G1 phase. Consistent with these observations, Pon2-/- B-ALL cells had a substantially reduced proliferation rate and expressed Arf and p21 checkpoint proteins at substantially increased levels compared to Pon2+/+ ALL cells. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in mouse transplant experiments for BCR-ABL1 and NRASG12D B-ALL models. Pon2-deficiency substantially prolonged survival of recipient mice of BCR-ABL1 B-ALL cells (P=0.0001). Strikingly, NSG mice transplanted with Pon2-deficient NRASG12D B-ALL cells did not develop any leukemia and survived for indefinite periods of time. Based on engineered expression of Cas9 and PON2-specific cRNAs, genetic deletion of PON2 replicated these findings in patient-derived B-ALL xenografts (PDX). Mechanism: Reflecting PAX5 and IKZF1 function, B-lymphoid cells exhibit transcriptional repression of glucose transport (e.g. repression of INSR, GLUT1). Interestingly, deletion of PON2 decreased glucose uptake even further. Intracellular ATP levels in Ph+ ALL cells were drastically reduced and activated the AMPK energy-stress sensor in both murine BCR-ABL1 and NRASG12D B-ALL models and human Ph+ B-ALL cells. Reconstitution of PON2 restored ATP production as early as 3 hours after PON2 induction. Mechanistically, PON2 was previously identified as an inhibitor of STOM and GLUT1 interactions. By disrupting STOM: GLUT1 interactions, PON2 can restore glucose transport. STOM-knockdown in PON2-/- ALL cells replenished cellular ATP levels as did PON2 reconstitution. We conclude that PON2 serves a critical role in B-ALL by subverting the metabolic gatekeeper function of IKZF1 and PAX5 through interference with STOM:GLUT1 interactions. Therapeutic targeting of PON2: While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts. Hence, we tested the concept of leveraging PON2 lactonase activity in a prodrug-approach as a synthetic lethal in Pon2+ B-ALL cells. 3OC12 had profound cytotoxic effects in wildtype but not Pon2-/- B-ALL cells while reconstitution or overexpression of PON2 restored cytotoxic responses to 3OC12. Conclusion: We describe the previously unrecognized function of the detoxifying PON2 lactonase as a central factor in glucose and energy supply in B-ALL, hence subverting metabolic gatekeeper functions of PAX5 and IKZF1. In addition to identifying PON2 as the biomarker for for Ph+ and Ph-like ALL and outcome predictor for patients with B-ALL, our results indicate PON2 facilitate glucose uptake and ATP production via its interaction with STOM. PON2 protects B-ALL cells against glucose depletion and enables malignant growth. On the other hand, we demonstrate a targeted approach that leverages PON2-lactonase as a synthetic lethal to the lactone-prodrug 3OC12. Disclosures No relevant conflicts of interest to declare.

Autonomous Ca2+ Oscillations Reflect Oncogenic Signaling in B-ALL Cells

Background: Engagement of the B-cell receptor (BCR) results in Ca2+ flux and is linked to B-cell survival based on store-operated Ca2+ entry (SOCE), which is triggered by ORAI1 and stromal interaction molecule-1 (STIM1). While normal B-cells release Ca2+ only in response to external stimuli (e.g. BCR-engagement), we recently observed that transformed B-cells exhibit autonomous oscillatory Ca2+ signals that are linked to oncogene activity. Studying a novel dual biosensor system to concurrently measure Ca2+ fluctuations and oncogenic kinase activity in the same B-ALL cells, we discovered that oncogenic kinase- and Ca2+-signals alternate between On- and Off-phases, during which kinase and Ca2+ signals are mutually exclusive. Results: Consistent with a scenario in which autonomous Ca2+-signaling is linked to oncogenic signaling, we observed that high mRNA levels of Orai1 and Stim1 in patients with B-ALL were linked to poor clinical outcomes. To study the role of SOCE and its effectors Orai1 and Stim1 in oncogene signaling, we developed mouse models for inducible deletion of Orai1 and Stim1/2 in BCR-ABL1- and NRASG12D-driven B-ALL. Tamoxifen-inducible activation of Cre in Orai1fl/fl and Stim1/2 fl/fl BCR-ABL1- and NRASG12D-driven B-ALL cells induced excision of both SOCE-mediators and induced near-complete loss of Ca2+-signaling competence. While treatment with the Ca2+ pump inhibitor, thapsigargin, elicited a strong SOCE signal, Ca2+-signaling was entirely muted upon Orai1-deletion, while deletion of Stim1 diminished and substantially delayed the residual Ca2+-signal. Studying autonomous Ca2+-signaling activity in B-ALL cells, Orai1-deletion resulted in a complete loss of oscillatory Ca2+-signaling, while deletion of Stim1/2 distorted the morphology of the Ca2+-signals with lowered amplitude and greatly extended signal duration. While both Orai1 and Stim1 contribute to SOCE, these observations highlight distinct roles of the two molecules in autonomous Ca2+ oscillation. For both BCR-ABL1- and NRASG12D-driven B-ALL, ablation of both Orai1- and Stim1/2 had profound effects on cell viability and the ability to form leukemic colonies. These results are consistent with the hypothesis that SOCE and autonomous Ca2+-oscillations are essential for oncogenic signaling in B-ALL and thus enable colony formation, proliferation and survival. To elucidate a functional link between autonomous Ca2+-oscillations and oncogenic signaling in B-ALL, we studied activation of Nfatc1, a central regulator of oncogene signaling and frequency-dependent decoder of Ca2+-oscillations. Upon Cre-mediated deletion of Orai1 and Stim1/2, however, Nfatc1 no longer translocated to the nucleus and was subject to increased proteasomal degradation. Establishing a functional link between Nfatc1-activation and survival and proliferation signaling in B-ALL cells, inducible activation of Nfatc1-deletion in BCR-ABL1-transformed Nfatc1flfl B-ALL cells phenocopied the effects observed with genetic deletion of Orai1 and Stim1/2. Interestingly, the NFATC1-inhibitor INCA6 exhibited strong cytotoxic responses in human B-ALL cells with strong Ca2+-oscillations, while Hodgkin's lymphoma and myeloma cells, lacking Ca2+-oscillations, were resistant to INCA6. To directly determine how oncogenic signaling impinges on Ca2+-oscillations, we developed a dual biosensor to concurrently measure Ca2+ fluctuations (RCaMP; red) and oncogenic BCR-ABL1 kinase activity (CRKL-phosphorylation; green) in the same B-ALL cells. Simultaneous measurement of Ca2+ (red) and kinase (green) activity revealed that B-ALL cells continuously alternated between Red-only and Green-only, suggesting that kinase and Ca2+ signals both oscillate in a mutually exclusive manner. Conclusions: Our findings show central roles of SOCE-effector genes, Orai1 and Stim1/2, in oncogenic signaling of B-ALL cells and activation of Nfatc1 as a driver of proliferation and survival signaling. Engineering of a dual biosensor for concurrent measurement of Ca2+ and oncogenic kinase activity revealed that B-ALL cells continuously alternate between Ca2+ and oncogenic kinase activity, with Ca2+-peaks marking the off-state of the oncogenic kinase. Disclosures No relevant conflicts of interest to declare.