B-cell Precursor Acute Lymphoblastic Leukemia Cells Research Articles

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Central role of the mTORC1 pathway in glucocorticoid activity against B-ALL cells

AbstractGlucocorticoids (GCs), such as dexamethasone and prednisone, are crucial components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapies. However, the molecular basis of GC-induced cell death remains elusive. Here, we show that GC suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling and that, conversely, oncogenic activation of mTORC1 confers resistance to GCs. Our genome-wide CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) dropout screens reveal that depletion of components of either the gap activity toward Rags 1 or tuberous sclerosis complexes, both negative regulators of mTORC1 signaling, significantly attenuates B-ALL cell sensitivity to dexamethasone. Dexamethasone primarily induces B-ALL cell death by downregulating mTORC1 activity, thus promoting autophagy and impairing protein synthesis. Dexamethasone treatment failed to suppress mTORC1 activity in B-ALL cells expressing mutant GC receptors lacking DNA-binding capacity, suggesting that dexamethasone transcriptionally represses mTORC1 activity. RNA-sequencing analysis identified multiple dexamethasone target genes that negatively regulate mTORC1 activity. Our findings suggest that GC sensitivity is significantly influenced by oncogenic stimuli and/or growth factors that activate the PI3K-AKT-mTORC1 pathway. This is consistent with the frequent GC resistance found in Ph and Ph-like ALLs.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Long Non-Coding RNA RP11-252C15.1 Is a Potential Biomarker of Prognosis and Hallmark for Leukemogenesis in Children with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Improved understanding of the prognostic biomarkers associated with childhood acute lymphoblastic leukemia (ALL) is needed for accurate risk group stratification. This study aimed to identify potential long non-coding RNA (lncRNA) markers and evaluate their prognostic value in children with ALL. Methods: We selected 50 children with newly diagnosed ALL and 20 age-matched patients with idiopathic immune thrombocytopenia (controls). RNA sequencing was performed to identify differentially expressed lncRNAs between the ALL and control groups. Correlation analysis was performed to determine the relationships between candidate lncRNAs, clinical features, and the risk of leukemogenesis. Results: A total of 1,019 differentially expressed lncRNAs were identified between the ALL and control groups. Reverse transcriptase (RT-qPCR) revealed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 were significantly upregulated in patients with ALL. Furthermore, correlation analysis showed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 represent potential predictors of leukemogenesis; however, only lncRNA RP11-252C15.1 was associated with clinical features and outcome in children with B-cell precursor ALL (BCP-ALL). In vitro experiments confirmed that lncRNA RP11-252C15.1 was significantly overexpressed in BCP-ALL cell lines and promoted proliferation and repressed apoptosis in MHH-CALL-3 cells. Conclusion: lncRNA RP11-252C15.1 is a potential oncogene in BCP-ALL pathogenesis and a prognostic biomarker in children with BCP-ALL.

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/β response in bone marrow-derived mesenchymal stroma.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance. Bone marrow-derived mesenchymal stromal cells (MSCs) mimic this protective environment in ex vivo co-cultures with leukemic cells obtained from children with newly diagnosed BCP-ALL. We examined the potential mechanisms of this protection by RNA sequencing of flowsorted MSCs after co-culture with BCP-ALL cells. Leukemic cells induced an interferon (IFN)-related gene signature in MSCs, which was partially dependent on direct cell-cell signaling. The signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1 positive ALL cells, as coculture of MSCs with healthy immune cells did not provoke a similar IFN signature. Leukemic cells and MSCs both secreted IFNα and IFNβ, but no IFNγ. In line, the IFN-gene signature was sensitive to blockade of IFNα/β signaling, but less to that of IFNγ. The viability of leukemic cells and level of resistance to three chemotherapeutic agents was not affected by interference with IFN signaling using selective IFNα/β inhibitors or silencing of IFN-related genes. Taken together, our data suggest that the leukemia-induced expression of IFNα/β-related genes by MSCs does not support survival of BCPALL cells but may serve a different role in the pathobiology of BCP-ALL.

Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.

The impact of an additional copy of chromosome 21 in B-cell precursor acute lymphoblastic leukemia.

A common finding in pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) is that chromosome 21 is never lost and an extra chromosome 21 is often gained. This implies an important role for chromosome 21 in the pathobiology of BCPALL, emphasized by the increased risk of BCPALL in children with Down syndrome. However, model systems of chromosome 21 gain are lacking. We therefore developed a BCPALL cell line (Nalm-6, DUX4-rearranged) with an additional chromosome 21 by means of microcell-mediated chromosome transfer. FISH, PCR, multiplex ligation-dependent probe amplification, and whole exome sequencing showed that an additional chromosome 21 was successfully transferred to the recipient cells. Transcription of some but not all genes on chromosome 21 was increased, indicating tight transcriptional regulation. Nalm-6 cells with an additional chromosome 21 proliferated slightly slower compared with parental Nalm-6 and sensitivity to induction chemotherapeutics was mildly increased. The extra copy of chromosome 21 did not confer sensitivity to targeted signaling inhibitors. In conclusion, a BCPALL cell line with an additional human chromosome 21 was developed, validated, and subjected to functional studies, which showed a minor but potentially relevant effect in vitro. This cell line offers the possibility to study further the role of chromosome 21 in ALL.

Genetic Subtype Driven Changes in Bone Marrow Stromal Cells of Children with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction We previously showed that B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance [1-3]. Bone marrow-derived mesenchymal stromal cells (MSCs) are a major component of the bone marrow microenvironment . Ex vivo,these cells provide a survival benefit to co-cultured leukemic cells and induce resistance to chemotherapeutic drugs [1, 2]. We previously showed that leukemic cells re-sensitize to chemotherapeutics ex vivo when the interaction with MSCs is disrupted [2]. Results We here report that patients' BCP-ALL cells induce an Interferon (IFN)-related gene signature in patients'-derived MSCs; a.o. IFI6, MX1, and OAS3 were 4.3 to 7.7-fold upregulated in MSCs flow-sorted after co-culture with BCP-ALL cells compared to the same MSCs flow-sorted after mono-culture . BCP-ALL samples (n=15) with an ETV6-RUNX1 translocation (5 out of 8 samples) were the most potent inducers of the IFN-related gene expression signature in MSCs compared to B-other BCP-ALL (1 out of 6 samples), with IFI6 3.0-fold (p=0.03), MX1 2.6-fold(p=0.05) and OAS3 2.1-fold (p=0.04) higher in MSCs after co-culture with ETV6-RUNX1 BCP-ALL cells. Similarly, an ETV6-RUNX1cell line (REH) also induced this IFN-response more strongly in MSCs compared to cell lines representing other BCP-ALL subtypes (SUPB15, MUTZ5, RCH-ACV and NALM6). Addition of specific inhibitors to these ex vivo BCP-ALL and MSCs cultures, revealed that the ETV6-RUNX1 induced IFN signature was dependent on IFNα/β signaling, whereas no such effect was seen for inhibitors of IFNγ signaling. Additionally, inhibition of direct contact between BCP-ALL cells and MSCs also negatively affected the expression of some IFN responsive genes in MSCs ( CXCL10 3.3-fold, p=0.02, IFI44L 1.57-fold, p=0.01 and IFITM1 1.5-fold, p=0.005) while other genes remained unaltered, suggesting that the transcriptional changes in MSCs are driven by close contact with BCP-ALL cells and not by soluble factors released by these cells or those being part of the culture medium. Normal immune cells including various populations of T-cells, monocytes, NK cells and dendritic cells did not alter the expression levels of the IFN-related genes in MSCs. We also observed that, while MSCs provide a survival benefit to (ETV6-RUNX1) BCP-ALL cells and induce resistance to chemotherapeutic drugs (prednisolone, daunorubicin, L-asparaginase) in these leukemic cells, this was not counteracted by inhibition of IFNα/β signaling. Conclusion Our data show that leukemic cells, more specifically ETV6-RUNX1 positive BCP-ALL cells, induce an IFNα/β signature in MSCs. Activation of this IFNα/β pathway in MSCs does not cause resistance of BCP-ALL cells to chemotherapeutic drugs and hence the IFNα/β activation may serve a different role, e.g., affecting the migration (recruitment and/or repelling) of specific immune cells to the leukemic bone marrow niche. Our findings warrant further studies into the role of the BCP-ALL-induced IFNα/β response in chemotaxis of healthy immune cells to create a beneficial microenvironment for BCP-ALL cells. This knowledge might point to alternative, maybe less intensive ways to eradicate leukemic cells.

CNS Invasion of TCF3::PBX1+ Leukemia Cells Requires Upregulation of AP-1 Signaling As Revealed By Brain Organoid Model

Introduction: Involvement of the central nervous system (CNS) remains a challenge in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Currently, the investigation of CNS leukemia mechanisms relies heavily on 2D cell culture and mouse models. However, given the differences between human and murine CNS in cellular identity and architecture, it becomes crucial to explore alternative models to study CNS leukemia. Moreover, novel targets for diagnosis and treatment of CNS ALL are critically needed. Methods: In this study, we established a pioneering 3D co-culture model that combines human induced pluripotent stem cell (iPSC)-derived cerebral organoids with BCP-ALL cells. We developed new methods to extract data from our invasion assays by enhancing the visualization of 3D organoid images, enabling us to accurately measure the invasion depth of leukemia cells compared to healthy controls within the organoids relative to their surface. To gain further insights on leukemia cells invading the organoids compared to those in the non-invaded fraction, we conducted RNA sequencing and immunofluorescence staining. Subsequently, we validated these results in a BCP-ALL in vivo mouse model and in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases within a cohort of 100 BCP-ALL patients. Results: Our experiments demonstrated robust and deep engraftment of TCF3::PBX1+ leukemia cell lines and patient-derived xenograft (PDX) cells into cerebral organoids (within a 14-day of co-culture). In contrast, the engraftment of healthy human CD34+ hematopoietic stem and progenitor cells (HSPCs) was limited ( p < 0.05) as compared to leukemia cells. Utilizing the co-culture model, we successfully validated the targeting of CNS leukemia-relevant pathways, such as CD79a/Igα or CXCR4-SDF1, via genetic knockdown or blocking experiments using inhibitor, respectively, which reduced the invasion of BCP-ALL cells into the organoids. Of note, RNA sequencing and immunofluorescence staining analysis revealed a significant upregulation of members of the AP-1 transcription factor complex, namely FOS, FOSB, and JUN, in the organoid-invading cells. Furthermore, we found an enrichment of AP-1 pathway genes in PDX cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX BCP-ALL cells (n = 5), thereby supporting the critical role of AP-1 signaling in CNS disease (Figure 1A). In line with these findings, we observed significantly higher levels of the AP-1 gene JUN in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases (mean JUN expression: 633.4 ± 78.51 in CNS-negative vs 1142 ± 296.5 in CNS-positive) within a cohort of 100 BCP-ALL patients (Figure 1B). Summary: In summary, we present ALL co-culture systems with iPSC-derived cerebral organoids as a promising complementary model to investigate CNS involvement in BCP-ALL including therapeutic targeting approaches, and identified the AP-1 pathway as a marker of CNS disease in TCF3::PBX1+ BCP-ALL.

In Vitro Efficacy of Anti-HER2 CAR T-Cells in HER2 + BCP-ALL

Introduction: Around 30% of adults and 15% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse after a first line of treatment. The prognosis of these patients remains poor. Recent advances have involved targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T-lymphocytes (anti-CD19 CAR T-cells), which have produced very encouraging results in the high risk BCP-ALL setting(Maude et al. 2018). Nevertheless, half of these patients relapse after such therapies. In a third of cases, the disease no longer expresses the antigen previously targeted. New targets are therefore needed. HER2 is expressed in above one third of adult BCP-ALL (Chevallier et al. 2004) and 10% of childhood BCP-ALL (Camuset et al. 2021). HER2 expression is associated with chemoresistance and poor prognosis (Chevallier et al. 2004) (Camuset et al. 2021). The anti-HER2 antibody trastuzumab was prospectively tested in adult patients with HER2+ BCP-ALL, but yielded disappointing results (Chevallier et al. 2012). Anti-HER2 CAR T-cells could be an interesting alternative. Indeed, they have shown encouraging preclinical efficacy in solid tumors expressing HER2/neu (Budi et al. 2022). The present work was designed to evaluate, in vitro, the sensitivity of HER2+ BCP-ALL to anti-HER2 CAR engineered Epstein Barr virus (EBV) specific T-cells. Methods: DMSO-frozen cell-samples from 6 HER2+ BCP-ALL patients were available for this study. Patient characteristics are shown in Table 1. Blast cells from patient samples were preferred to cell lines in order to be more representative of BCP-ALL biology. level of HER2 expression on BCP-ALL samples was determined by flow cytometry and compared to the HER2 expressing breast-cancer cell line BT474. Anti-EBV cytotoxic T lymphocytes (CTLs), armed with a trastuzumab-based scFv-CH2-CH3-IgG2A-FcεRIγ chimeric receptor as previously described (Clémenceau et al. 2015) were used for long-term cytoxicity assays. For 4 samples, non-transduced anti-EBV CTLs were used as controls. Cytotoxic activity was assessed at a 3-to-1 effector-to-target ratio, in a flow cytometry assay. Cells were harvested at H0, H4 and H24 and stained with a viability stain (VS780). Further staining with anti-CD22 or anti-CD24 (depending on the BCP-ALL immunophenotype) and anti-CD3 antibodies allowed to characterize blast cells and CAR T-cells respectively. The study was approved by the local review board and two alive patients provided informed consent. Results: The level of HER2 expression by the 6 HER2+ BCP-ALL samples was variable with a median relative fluorescence intensity ratio (RFI) of 9 (range 1.8 to 16). These levels of expression were lower than those of the BT474 cell line (RFI= 79). No BCP-ALL lysis was observed when cells were incubated with trastuzumab alone (not shown). When co-cultured with non-transduced anti-EBV CTLs (CAR negative) (n=4), a decrease of residual viable BCP-ALL cells was observed at variable levels (Figure 1A). The lysis observed at 24h was possibly due to allogeneic recognition since anti-EBV CTLs were not autologous to the leukemias tested. Conclusion: This study first reveals that, although CTLs with TCRs restricted to EBV antigens and therefore intrinsically low allo-specificity were used, CAR-independent lysis was observed in 24 hours co-culture with BCP-ALL cells. These results underline the need for cautious interpretation of cytotoxicity test results in an allogeneic setting. Secondly, these preliminary data suggest that an anti-HER2 CAR T-cell approach could be promising for BCP-ALL even with weak antigen expression. Anti-EBV CTLs have already been used as a platform for CAR-engineered T-cells (Rossig et al. 2017). Should the cytotoxic activity of allogeneic EBV-CTL be proven to be specifically directed against BCP-ALL, the synergy between TCR and anti-HER2-CAR might be beneficial, and adoptive transfer of anti-HER2 CAR EBV CTLs could be a promising therapeutic option for patients with HER2+ BCP-ALL. * This research is funded by the non-profit organization Etoile de Martin/SFCE.

CD38 Is a Functional Partner of CD9 in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: We previously showed that CD9 was expressed in ~80% of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases and its positivity was associated with an inferior relapse-free survival (Leung et al, Leukemia, 2020), suggesting its oncogenic role in this disease entity. Yet, the mechanisms underlying how CD9 affects leukemia progression remain completely unknown. As a prototypic member of the tetraspanin superfamily proteins, CD9 is able to organize with transmembrane or intracellular proteins to form the “tetraspanin web” to exert its modulatory functions. In BCP-ALL, though there has been a standalone study reporting 28 CD9-binding proteins in NALM-6 cells, the results are unlikely representative of the entire disease spectrum due to tumor heterogeneity. Here, we perform an in-depth proteomic study to dissect the CD9 web composition and downstream protein partners to drive disease progression in BCP-ALL. Methods: Immunoprecipitation coupled mass spectrometry (IP-MS) was performed to map the overall composition of CD9 web in five CD9 + BCP-ALL cell lines with distinct cytogenetic backgrounds, including 697 ( TCF3-PBX1+), BV-173 ( BCR-ABL1+), HAL-01 ( TCF3-HLF+), KASUMI-2 ( TCF3-PBX1+) and RS4;11 ( KMT2A-AFF1+). Overlapping protein partners were then validated by IP-Western blot (IP-WB) in 13 patient samples spanning major BCP-ALL subtypes. Shortlisted CD9 partners were further characterized by antibody blockade and CRISPR/Cas9 loss-of-function experiments to evaluate their impact on leukemia proliferation in vitro and leukemia progression in vivo, coupled with enzymatic and phenotypic assays to evaluate the direct impact of CD9 on partner functions. Results: A total of 278 CD9-binding proteins comprising 24 known and 254 novel partners were identified by IP-MS. Venn analysis revealed a marked heterogeneity of CD9-binding proteins across leukemia subtypes, with only 10 being intersected representing the “universal CD9 partners” in BCP-ALL. These proteins consisted of 5 established CD9 partners: ADAM10, CD19, CD38, CD81 and IGSF8 as well as 5 novel partners: ANXA11, HBG2, TMEM109, SLC44A1 and STXBP3. Two universal CD9 partners, namely CD38 and CD81, were consistently detected in all pediatric BCP-ALL samples. Neutralizing antibodies against CD9, CD38 or CD81 unanimously inhibited proliferation of 697 cells in vitro ( P<0.05). However, in the background of CD9 neutralization, CD81, but not CD38, offered an additional impact on leukemia suppression ( P<0.05). Likewise, CD38 knockout cells treated with CD9 antibody did not appear to inhibit cell proliferation in an additive manner. Concordantly, in the 697 xenografts, CD9 or CD38 antibodies alone prolonged animal survival by 2.1- and 1.6-fold, but combinational treatment did not produce the projected additive effect (2.5-fold only). In addition, CD9 neutralization significantly reduced the enzymatic activity of CD38 by 52.5% ( P<0.05) without altering its expression. Conclusions: This work presents the composition of the CD9 protein web in BCP-ALL, revealing homogenous and heterogeneous partners across the leukemia subtypes. We also provide definitive evidence showing that CD9 binds to and regulates CD38 activity to mediate leukemia progression, representing the first universal protein partner being identified to have a functional consequence in BCP-ALL, thereby providing a mechanistic basis to support the development of CD9-targeted therapies for this life-threatening malignancy.

The Topoisomerase-I Inhibitor Gimatecan Exhibits Potent and Selective Activities Against B-Cell Precursor Acute Lymphoblastic Leukemia with a Favorable Cardiotoxicity Profile

Topoisomerase-II inhibitors such as daunorubicin have long been the standard chemotherapeutic agents for managing acute leukemias. Yet, severe complications could be resulted from their myelosuppressive and cardiotoxic nature. Here, we report preclinical evidence showing the exceptional activities of gimatecan, an orally available topoisomerase-I inhibitor currently being investigated in Phase II trials for solid cancers, on hematologic malignancies. Activity screening on leukemia cell lines revealed a substantially higher potency of gimatecan than frontline chemotherapeutics against B-cell precursor acute lymphoblastic leukemia (BCP-ALL) driven by on-target depletion of topoisomerase-I. Gimatecan had a median IC50 of 0.9 nM against BCP-ALL with profound selectivity over acute myeloid leukemia (AML, 3.6-fold, P< 0.01) or hematopoietic stem/progenitor cells (HSPCs, 75-fold, P< 0.001). In contrast, the four standard chemotherapeutics (cytarabine, daunorubicin, dexamethasone and vincristine) exhibited much higher median IC50s against BCP-ALL (18-2,201 nM). Following single-agent gimatecan treatment, complete dissolution of medullary and extramedullary leukemia (CNS and testis) by >95% inhibition concomitant with markedly extended survival (1.2 to 3.3-fold, P< 0.01) were demonstrated in animals grafted with cytogenetically distinct lymphoid leukemia cell lines (Reh, ETV6-RUNX1+; 697, TCF3-PBX1+; RS4;11, KMT2A-AFF1+; HAL-01, TCF3-HLF+) and in patient-derived xenografts from three cases of highly resistant BCP-ALL failing salvage chemotherapy or upfront immunotherapies. Splenocytes isolated from NSG mice relapsed from gimatecan treatment demonstrated recurrent drug sensitivity ex vivo comparing with vehicle controls and that gimatecan reinduction offered additional survival benefits in the xenograft model ( P< 0.05). The therapeutic efficacy was also observed at varied initial tumor load, together suggesting its potential for reinduction and flexibility on the administration schedule. In NSG mice reconstituted with cord blood HSPCs, gimatecan displayed a mild but transient suppression on normal hematopoiesis without bias on lineage output (63% reduction at 6 weeks, P=0.002; not statistically significant at 14 weeks). Importantly, gimatecan did not affect the viability of human iPSC-derived cardiomyocytes (hiPSC-CM) even at the highest tested dose (1,000 nM) as opposed to daunorubicin (IC50: 130 nM). Concordantly, no noticeable signs of DNA damage (by γ-H2AX staining) or apoptotic events (by enumeration of condensed nuclei) on hiPSC-CMs were observed after gimatecan exposure at doses at or above its Cmax (~130 nM). The gross structure of heart tissues harvested from gimatecan-treated mice was also intact without signs of cardiotoxicity at the dose effective to suppress leukemia (0.2 mg/kg). When compared with malignant B-lymphoblasts, there was a declined expression of topoisomerase-I in HSPCs (0.63-fold, P=0.025) and cardiomyocytes (0.24-fold, P<0.001), potentially explaining its selectivity against BCP-ALL. Mechanistically, gimatecan induced time- and dose-dependent cell death via intrinsic apoptosis coupled with G2/M cell cycle arrest. RNA-seq analysis of gimatecan-treated BCP-ALL cells showed activation of the tumor suppressive p53 pathway and suppression of the MYC pathway. Phosphorylation of p53 and appearance of DNA damage markers, including CHK1/2, PARP and H2AX, were robustly detected upon gimatecan treatment. In short, gimatecan possesses remarkable anti-leukemia activities and tolerable toxicity profiles compared to counterpart topoisomerase-II inhibitor. This study, therefore, puts forward a new agent for BCP-ALL and points towards randomized clinical trials to realize its potential on acute leukemias.

Synergistic Anti-Cancer Effects of Second-Generation Proteasome Inhibitor Carfilzomib with Doxorubicin and Dexamethasone Via p53-Mediated Apoptosis in Pre-B Acute Lymphoblastic Leukemia Cells

Background: The ubiquitin-proteasome system (UPS) plays a crucial role in regulating the levels and functions of a large number of proteins in the cell, which are important for cancer cell growth and survival. The proteasome is highly activated in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), which is the most common malignancy in children. The attempt to inhibit proteasome as a therapeutic strategy has been successful for some malignancies.
 Materials and Methods: In this experimental study, human BCP-ALL cell lines NALM-6 and SUP-B15 were treated with carfilzomib with and without the chemotherapeutic agents. The XTT assay evaluated the viability of the cells. Cell cycle analysis and apoptosis assay were assessed by flow cytometry. RQ-PCR and western blotting evaluated the expression of pro-/anti-apoptotic signals. A drug combination study for synergistic or additive effects of carfilzomib with doxorubicin or dexamethasone was performed.
 Results: We observed that carfilzomib alone induced G2/M cell cycle arrest and caspase-dependent apoptosis in the human BCP-ALL cells (NALM-6 and SUP-B15). Gene and protein expression analysis indicated the upregulation of pro-apoptotic as well as downregulation of the cell survival and proliferative signals (P-value<0.05). The synergy of carfilzomib with doxorubicin or dexamethasone was revealed in BCP-ALL cells.
 Conclusion: Our results indicated that proteasome inhibition induces p53-mediated apoptosis in BCP-ALL cells. Since carfilzomib has a synergistic effect with anti-leukemic agents doxorubicin and dexamethasone in BCP-ALL cells, this combined-modality approach might be befitting for patients who do not respond well to conventional chemotherapy.

A Genome-Wide CRISPR-Cas9 Screen Reveals GATOR1 Complex Is a Critical Regulator of Glucocorticoid Sensitivity in B-Cell Precursor Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are essential components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapy. Upon GC binding, GC receptor (GR) translocates to the nucleus and activates GR target genes. While GCs reportedly induce cell death via apoptosis and/or cell cycle arrest, their molecular mechanisms remain largely unknown. To identify molecules/pathways governing GC sensitivity in B-ALL cells, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of dexamethasone (Dex) using two human B-ALL lines (NALM-6 and MUTZ-5). To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled highly efficient transduction of human B cells (Sasaki et al. Blood 2022). We identified a total of 90 and 46 genes, whose single-guide RNAs (sgRNAs) were significantly enriched upon Dex treatment in NALM-6 and MUTZ-5, respectively. A total of 15 genes were identified in both lines, and among them included those previously implicated in GR resistance, such as NR3C1 and TBL1XR1. Strikingly, sgRNAs targeting DEPDC5, NPRL2 and NPRL3, each encoding components of the GATOR1 complex, a negative regulator of mTORC1, were significantly enriched in the presence of Dex in both lines. To validate these findings, we depleted each gene via lentivirus-based E2-Crimson-tagged sgRNA and assessed effects on cell growth over 11 days with or without Dex. Cells expressing sgRNA targeting either DEPDC5, NPRL2 or NPRL3, but not those transduced with empty vector, demonstrated a proliferative advantage relative to non-transduced cells in the presence of Dex. Importantly, Dex treatment significantly reduced phospho-P70S6K levels, a readout of the mTOR pathway activation, in GATOR1-intact cells, but not in NPRL2 or DEPDC5 knockout cells, suggesting that Dex suppresses mTORC1-mediated growth signals via the GATOR1 complex. We next asked whether Dex/GATOR1-mediated mTORC1 suppression is mediated by transcriptional activity of GR. To do so, we established a NALM-6 line deficient in endogenous GR in which a GR mutant lacking DNA binding capacity was exogenously expressed. Dex treatment did not reduce phospho-P70S6K levels in these cells, suggesting that the Dex/GATOR1-mediated mTORC1 suppression seen in GATOR1-intact cells primarily depends on transcriptional capacity of GR. Given that loss-of-function mutations of DEPDC5 were reported in therapy-refractory B-ALL cases and that GATOR1 complex reportedly functions in amino acid metabolisms, our findings suggest a potential link between GC sensitivity and amino acid metabolism in B-ALL cells.

Histone Deacetylase Inhibition Restores Therapy Response in TP53mutated Acute Lymphoblastic Leukemia to Chemotherapy

Background: Although TP53 is the most frequently mutated gene in cancer, in Acute Lymphoblastic leukemia (ALL), mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse TP53 aberrations are found in about 12% of the patients and predict a very poor outcome. As a consequence, relapsed TP53 deleted ALL is now classified as 'very high risk, together with t(1;19) and t(17;19) ALL. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance not only to classical chemotherapeutics, but also newly introduced immunotherapies such as bi-specific T cell engagers such as Blinatumomab or CAR-T cell therapies. Therefore, there is an urgent clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods: Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wildtype and knockout models in the TP53WT B-cell precursor ALL (BCP-ALL) cell lines Nalm6 and RCH-ACV. After confirming the presence of TP53 deletions, we treated these isogenic models with a panel of treatment protocol drugs, demonstrating that TP53 loss negatively affects response to therapy to most contemporary drugs used in the treatment of relapsed ALL. To identify drug combinations that may reverse TP53 mediated therapy resistance, we assessed the viability of these isogenic models in a drug screen containing 198 (pre)clinical drugs. The most interesting targets were subsequently combined with the previously tested treatment protocol drugs to determine potential synergies. Effects on cell viability were determined by amine exposure and MTT assay and confirmed by measuring PARP cleavage. Additionally, positive drug interactions were validated in vivo using immunocompromised mice engrafted with RCH-ACV p53KO cells expressing a luciferase reporter gene. Results: We observed that loss of p53 protects BCP-ALL cell lines Nalm6 and RCHACV from drug-induced apoptosis, leading to increased resistance to all drugs used currently in the treatment of relapsed ALL, except for Asparaginase. In a subsequent drug screen, we found that the histone deacetylase (HDAC) 1/2 inhibitor romidepsin shows strong single agent activity at single nanomolar concentrations. Moreover, romidepsin was found to strongly synergize with contemporary ALL treatment drugs in TP53KO and TP53WT cells (Figure 1A). This effect was strongest in TP53KO cells, effectively reversing therapy resistance induced by TP53 loss. Gene expression profiling suggest that romidepsin may lead to reactivation of TP53 target genes in response to cellular stress, even in the absence of functional p53. Finally, preliminary mouse experiments using TP53 deleted RCH-ACV cells showed that romidepsin improves the response to cytarabine in TP53 aberrant leukemia in vivo (Figure 1B) without signs of added toxicity. A mouse trial testing a potential synergy between romidepsin and cytarabine using TP53 deleted patient derived xenografts (PDX) is currently underway. Conclusions: Loss of p53 protects BCP-ALL cells from currently used (chemo)therapy strategies. Romidepsin can restore response to therapy in TP53 deleted ALL, presumably by reactivation of p53 target genes in response to cellular stress. Therefore, romidepsin may be used to improve therapy response in relapsed TP53 deleted ALL. Since romidepsin already has FDA approval for use in other hematological malignancies, our findings may be rapidly translated into clinical practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow.

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon shortterm chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.

Association between Dysfunction of the Nucleolar Stress Response and Multidrug Resistance in Pediatric Acute Lymphoblastic Leukemia.

Simple SummaryPediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is one of the mechanisms that activate P53 by ribosomal protein L11 (RPL11). We hypothesized that the lack of nucleolar stress response is related to chemoresistance and relapse in some pediatric BCP-ALL cases. We revealed that clinical BCP-ALL therapeutics, such as 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine, induced the nucleolar stress response, and its treatment susceptibility was dependent on the nucleolar stress response. Furthermore, we observed decreased RPL11 expression at relapse in seven children with BCP-ALL in comparison to that at onset. Our findings provide new insights into the anti-leukemia mechanism in BCP-ALL and multidrug resistance and relapse via the nucleolar stress response, suggesting that the nucleolar stress response may be a potential therapeutic strategy to predict chemosensitivity and improve chemoresistance in pediatric BCP-ALL.Approximately 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is a P53-activating mechanism via MDM2 inhibition by ribosomal protein L11 (RPL11). We analyzed the role of the nucleolar stress response using BCP-ALL cell lines and patient samples by drug sensitivity tests, Western blotting, and reverse transcription polymerase chain reaction. We revealed that the nucleolar stress response works properly in TP53 wild-type human BCP-ALL cell lines. Next, we found that 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine had anti-leukemic effects via the nucleolar stress response within BCP-ALL treatment. Comparing the samples at onset and relapse in children with BCP-ALL, RPL11 mRNA expression decreased at relapse in seven of nine cases. Furthermore, leukemia cells with relapse acquired resistance to these four drugs and suppressed P53 and RPL11 expression. Our findings suggest that the nucleolar stress response is a novel anti-leukemia mechanism in BCP-ALL. As these four drugs are key therapeutics for BCP-ALL treatment, dysfunction of the nucleolar stress response may be related to clinical relapse or refractoriness. Nucleolar stress response may be a target to predict and improve the chemotherapy effect for pediatric BCP-ALL.

Normal and Aberrant TALE-Class Homeobox Gene Activities in Pro-B-Cells and B-Cell Precursor Acute Lymphoblastic Leukemia.

Homeobox genes encode transcription factors regulating basic developmental processes. They are arranged according to sequence similarities of their conserved homeobox in 11 classes, including TALE. Recently, we have reported the so-called TALE-code. This gene signature describes physiological expression patterns of all active TALE-class homeobox genes in the course of hematopoiesis. The TALE-code allows the evaluation of deregulated TALE homeobox genes in leukemia/lymphoma. Here, we extended the TALE-code to include the stages of pro-B-cells and pre-B-cells in early B-cell development. Detailed analysis of the complete lineage of B-cell differentiation revealed expression of TALE homeobox genes IRX1 and MEIS1 exclusively in pro-B-cells. Furthermore, we identified aberrant expression of IRX2, IRX3 and MEIS1 in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) which originates from early B-cell progenitors. The data showed correlated activities of deregulated TALE-class members with particular BCP-ALL subtype markers, namely IRX2 with TCF3/E2A-fusions, IRX3 with ETV6/TEL-fusions, and MEIS1 with KMT2A/MLL-fusions. These correlations were also detected in BCP-ALL cell lines which served as experimental models. We performed siRNA-mediated knockdown experiments and reporter gene assays to analyze regulatory connections. The results showed mutual activation of IRX1 and TCF3. In contrast, IRX2 directly repressed wild-type TCF3 while the fusion gene TCF3::PBX1 lost the binding site for IRX2 and remained unaltered. IRX3 mutually activated fusion gene ETV6::RUNX1 while activating itself by aberrantly expressed transcription factor KLF15. Finally, KMT2A activated MEIS1 which in turn supported the expression of IRX3. In summary, we revealed normal TALE homeobox gene expression in early B-cell development and identified aberrant activities of IRX2, IRX3 and MEIS1 in particular subtypes of BCP-ALL. Thus, these TALE homeobox genes may serve as novel diagnostic markers and therapeutic targets.

P339: BOOSTING THE EFFICACY OF CD20-TARGETING IMMUNOTHERAPY IN B CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: CD20 is a B cell-specific surface protein that appears at the stage of pre-B cells during B cell development. Currently, monoclonal antibodies (mAb) against CD20 are commonly used for immunotherapy of mature B-cell malignancies. B cell precursor acute lymphoblastic leukemia (BCP-ALL) originates from immature B cells in a bone marrow. Overall, the effects of treatment with high-dose multi-agent chemotherapy are good. However, for relapsed/refractory patients, novel treatment options (mainly immunotherapy-based) targeting B cell-specific antigens are already used in clinics and further optimized in preclinical models. Recent clinical trials conducted in Philadelphia (Ph) chromosome-negative BCP-ALL patients with CD20 expression revealed that the outcome of young adults may be improved by a combination of chemotherapy with rituximab, the anti-CD20 mAb. However, in most patients, the expression of CD20 on BCP-ALL blasts is heterogeneous and may be insufficient for effective treatment with CD20 mAb. Also, the regulation of CD20 in BCP-ALL is not well defined. Aims: The aim of this study is to identify drugs that are able to upregulate CD20 in BCP-ALL, in order to improve the efficacy of anti-CD20 mAbs in preclinical models of the disease. Methods: We tested the impact of 38 anti-cancer drugs used for the treatment of BCP-ALL as well as tested in clinical trials on the CD20 surface levels after 48hrs of incubation. For the in vitro studies, we employed cell lines and primary cells representing selected high-risk subtypes (Ph-positive, Ph-like, B-other). The levels of CD20 were determined by flow cytometry. Next, the potential of selected drugs to activate the anti-CD20 mAb, rituximab, was tested in a functional assay, Antibody-Dependent Cellular Cytotoxicity (ADCC) with Human Peripheral Blood Mononuclear Cells (PBMC) or primary NK cells, isolated from healthy donors. Results: Based on the initial screening of cell lines, we selected 10 agents which increased the level of CD20: three histone deacetylases inhibitors, one inhibitor of MDM2 protein (p53 signaling pathway), one apoptosis inducer, three kinase inhibitors, one mitosis inhibitor, and one antimetabolic agent. Importantly, we found that many tyrosine kinases inhibitors and some retinoids significantly decreased CD20 levels in BCP-ALL cell lines. Some of these findings were also confirmed in primograft BCP-ALL blasts co-cultured with OP9 murine stromal cells. Furthemore, we found that five out of ten drugs enhance antibody-dependent cytotoxicity of rituximab, in functional assay. Moreover, two of the tested drugs increased phagocytosis of primograft BCP-ALL cells by primary macrophages differentiated from human peripheral blood monocytes. Summary/Conclusion: In summary, we identified agents upregulating CD20 levels in BCP-ALL cells in vitro. More importantly, some of the selected drugs improved RTX-mediated effector mechanisms such as ADCC and immunophagocytosis. Further studies aiming to elucidate the mechanisms of CD20 upregulation and to validate our in vitro findings in murine models are ongoing.