B-cell Precursor Acute Lymphoblastic Leukemia Samples Research Articles

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.

RNA-seq–based miRNA signature as an independent predictor of relapse in pediatric B-cell acute lymphoblastic leukemia

AbstractAberrant micro-RNA (miRNA) expression profiles have been associated with disease progression and clinical outcome in pediatric cancers. However, few studies have analyzed genome-wide dysregulation of miRNAs and messenger RNAs (mRNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To identify novel prognostic factors, we comprehensively investigated miRNA and mRNA sequencing (miRNA-seq and mRNA-seq) data in pediatric BCP-ALL samples with poor outcome. We analyzed 180 patients, including 43 matched pairs at diagnosis and relapse. Consensus clustering of miRNA expression data revealed a distinct profile characterized by mainly downregulation of miRNAs (referred to as an miR-low cluster [MLC]). The MLC profile was not associated with any known genetic subgroups. Intriguingly, patients classified as MLC had significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 ×10−5). Furthermore, this poor prognosis was retained even in hyperdiploid ALL. This poor prognostic MLC profiling was confirmed in the validation cohort. Notably, non-MLC profiling at diagnosis (n = 9 of 23; Fisher exact test, P = .039) often changed into MLC profiling at relapse for the same patient. Integrated analysis of miRNA-seq and mRNA-seq data revealed that the transcriptional profile of MLC was characterized by enrichment of MYC target and oxidative phosphorylation genes, reduced intron retention, and low expression of DICER1. Thus, our miRNA-mRNA integration approach yielded a truly unbiased molecular stratification of pediatric BCP-ALL cases based on a novel prognostic miRNA signature, which may lead to better clinical outcomes.

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.

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.

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.

Acquired Venetoclax Resistance in an In Vivo Model of B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Altered Functions of Apoptosis Regulators

Deregulation of cell death pathways such as apoptosis is a hallmark of cancer and contributes 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 signaling. Inhibition of anti-apoptotic molecules of BCL-2 family cell death inhibitors has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to BCL-2 resulting in apoptosis induction and has shown clinical activity in hematologic malignancies, but primary and secondary resistance have also been described. In this study, we aimed to identify resistance mechanisms acquired during VEN treatment leading to therapy failure of BCL-2 inhibition. In order to mimic a clinical situation with consecutive treatment cycles, we employed a patient-derived human BCP-ALL xenograft mouse model with subsequent courses of VEN administration to ALL bearing recipients in vivo. First, we estimated VEN-sensitivities in a series of BCP-ALL PDX samples (N=30) by ex vivo titration of individual VEN-EC 50 values and identified a range of heterogeneous VEN-activities. A VEN-sensitive (low VEN-EC50) PDX-ALL sample with a pro-B ALL phenotype carrying a KMT2A:: MLL1 gene fusion was selected and transplanted onto immunodeficient NOD/SCID recipient mice. Upon leukemia manifestation (presence of 5% human ALL cells in peripheral blood), mice were either treated with VEN (100 mg/kg, two times five days) or vehicle control. Recipients were sacrificed upon onset of leukemia-related morbidity and ALL cells isolated from spleens were transplanted onto secondary recipients, which were again treated with VEN or vehicle. ALL cells isolated from secondary recipients were retransferred onto tertiary mice and again exposed to in vivo VEN-treatment providing a total of three subsequent transplantation and in vivo VEN-therapy cycles. Assessing VEN sensitivities of the ALL cells initially transplanted and isolated after VEN-treatment cycles by using an ex vivo cytochrome c release assay showed increasing VEN-resistance. Accordingly, comparison of VEN to control in vivo treated cells showed significantly decreased sensitivities over treatment cycles. Importantly, also the time periods between treatment and leukemia re-occurrence in mice indicating decreasing VEN-sensitivity became shorter with every additional in vivo therapy course. Paired RNA- seq analysis revealed that transcript levels of the pro-apoptotic molecules BAX and BIM were significantly downregulated in VEN-exposed/resistant ALL as compared to vehicle control treated cells. Basal BH3 profiling analysis showed impaired apoptotic priming (significantly lower cytochrome c release after BIM and PUMA peptide exposure) and decreased dependence on anti-apoptotic BCL-2 and MCL-1 indicating a shift from a pro-apoptotic to a pro-survival state. This shift was also reflected in an e x vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC 50 1.5 vs. 2.2 µM, A-1331852 EC 50 9.3 vs. 15.3 µM). In line with the low BAX levels in ALL cells with acquired VEN-resistance, PDX BCP-ALL samples with intrinsic VEN resistance also showed lower BAX protein expression. Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age.

Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient's sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.

Clinical Significance of IGH Clonality Analysis with RNA Sequencing in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common pediatric malignancy. Using minimal residual disease (MRD) and genetic subtypes, risk stratification algorithms have considerably improved clinical outcomes. However, despite being in a better prognostic group, a non-negligible number of relapses occur, and prognostic markers for these patients are required for more effective treatment strategies. RNA sequencing (RNA-seq) is a useful tool for detecting patient-specific immunoglobulin heavy chain (IGH) junctional sequences (Li et al., Leukemia. 2020), which can be informative for evaluating the nature of leukemic clones. Here, we analyzed IGH clonality of pediatric BCP-ALL using RNA-seq data and revealed its validity for evaluating disease characteristics and impact on prognosis. Methods: We analyzed RNA-seq data of 136 pediatric BCP-ALL samples at diagnosis and 46 samples at relapse from the TARGET ALL Expansion Phase 2 cohort. Using MiXCR (Bolotin et al., Nat Methods 2015) and Vidjil (Duez et al., PLoS One 2016), we detected IGH complete VDJ and incomplete DJ rearrangements. Then, we estimated the expected number of IGH rearrangements corresponding to each read count (E-value) by power-law model. Finally, we identified leukemic "disease clones” with E-values less than 0.01 (Fig. 1a). Results: First, we detected a total of 55,518 IGH clones, of which 48,555 were complete rearrangements and 6,963 were incomplete. In total, 351 disease clones (median 2, range 0-8 per sample) were identified by power-law model: 193 clones were complete rearrangements, and 158 clones were incomplete. Interestingly, the numbers and compositions (complete or incomplete rearrangements) of disease clones varied significantly between samples. Thus, we defined three clonality patterns and classified each sample based on identified disease clones: "Undetectable disease clones (UDC)” if the sample had no disease clone (n=18); "Incomplete Disease Clones (IDC)” if there were one or more disease clones with incomplete rearrangements (n=72); and "Complete Disease Clones (CDC)” if all disease clones of that sample have complete rearrangements (n=46). Gene Set Enrichment Analysis (GSEA) indicated a strong enrichment for hematopoietic stem cell (HSC) and multipotent progenitor (MPP) signatures in UDC and pro-B cell signatures in IDC. Similarly, CIBERSORTx (Newman et al., Nat Biotechnol 2019) showed higher proportions of HSCs, MPPs, and pre-pro B cells in UDC and pro-B cells in IDC. Furthermore, there was a significant enrichment in pathways promoting cell cycle progression in IDC. Of note, in high hyperdiploid (HHD) cases, which are typically associated with a favorable prognosis, event-free survival (EFS) was significantly lower in UDC and IDC (p=0.01) (Fig. 1b). Moreover, even in MRD-negative HHD patients, those with UDC and IDC had significantly worse EFS (p=0.01) and overall survival (p=0.03) (Fig. 1b). Finally, we compared the primary and relapse clones in 46 patients. As a result, 37 patients had one or more disease clones at diagnosis, and 29 (78%) had these primary disease clones at relapse. On the other hand, 9 (24%) patients showed relapse-specific disease clones, which should lead to relapse in these cases. The other 9 primary patients with UDC had no disease clone also at relapse. Discussions: We identified three clonality patterns, UDC, IDC, and CDC based on RNA-seq analysis of the IGH rearrangement status. According to the findings of GSEA and CIBERSORTx, leukemic cells of UDC, IDC, and CDC patients have nature of progenitors to pre-proB cells, pro-B cells, and more mature cells, respectively. These findings are consistent with the knowledge that IGH rearrangement occurs during pro-B to pre-B maturation in normal B-cells. Patients with immature clonality status, UDC and IDC, were associated with poor prognosis in high hyperdiploid subtype and could be a potential prognostic marker in MRD-negative patients. In addition, we revealed that primary disease clones contributed to relapse in 78% of cases, while relapse-specific clones were implicated in relapse in 24% of cases. Additionally, UDC clonality pattern was carried over at relapse. These findings shed light on the mechanisms of leukemic clonal evolution and relapse, and suggest that the status of IGH rearrangement may serve as a novel prognostic marker in BCP-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IKZF1 DELETIONS IN B-ALL: FROM GENETIC BASIS TO DIAGNOSTIC ENHANCEMENT

Because IKZF1 deletions are associated with an increased risk of relapse in patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), the development of methods for rapid and accurate detection of this alteration has a great clinical impact. Although multiplex ligation-dependent probe amplification (MLPA) has been widely used for the evaluation of IKZF1 deletions, multiplex (M)-PCR is a valuable tool for the validation of those IKZF1 deletions. However, this method is still limited to detect recurrent intragenic deletions (Δ2-3, Δ2-7, Δ2-8, Δ4-7, Δ4-8). Therefore, here we evaluate the breakpoint map of IKZF1 deletions with a special focus on non-recurrent deletions to update methodologies for the diagnosis of this genetic alteration. Moreover, we explore the genetic features underlying those IKZF1 deletions to infer its molecular pathogenesis. This study included four international cohorts of pediatric and adult patients with B-ALL. IKZF1 deletions were identified by MLPA (P335 and P202 assays) and M-PCR. Non-recurrent deletions were verified by next-generation sequencing using the Illumina DNA Prep with Enrichment assay. We also revised previously published data regarding IKZF1 deletions in B-ALL for compiling breakpoint sequence information of these deletions. Sequence data from our cohorts and literature were used in further analyses. After identifying the breakpoint clusters, we performed an agnostic motif search using MEME and annotated the presence of filler DNA or microhomologies at the junctions of IKZF1 deletions. A total of 1,472 B-ALL samples were screened, and IKZF1 deletions were found in 16% of these patients. Although non-recurrent deletions were associated with 1% of the total cohort, it represents 8% of all IKZF1 deletions. The analysis of breakpoint sequences at DNA level revealed that non-recurrent IKZF1 deletions comprises miscellaneous alterations, but the majority of them encompases Δ1 and Δ1-3. Considering that IKZF1 exon 1 deletions are more difficult to reliably detect by MLPA due to the high GC content (>50%) of this region, we mapped several exon 1 deletions at DNA level and identified one commonly deleted region which is ideal for designing MLPA probes. Next, we searched for motifs, additional nucleotides/microhomologies associated with IKZF1 deletion sequences. We verified an enrichment of the 5’-CACACAGTG-3’sequence within 21 out of 24 breakpoint clusters of IKZF1 deletion (E-value: 2.9e-008). Additionally, the filler DNA within the junctions of IKZF1 deletions presented a higher GC nucleotide content at these clusters as compared to the deletions outside of them (71% vs. 56%). This study provides the genetic landscape of IKZF1 deletions in B-ALL. Although rare types of deletion may be identified at the routine diagnosis, they should be considered after using adapted MLPA protocols, including additional probes within the common deleted region nearby IKZF1 exon 1. We also verified the occurrence of the heptamer sequence and an enrichment of GC nucleotides exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote most IKZF1 deletions, although rare types of alterations may be associated with another molecular mechanism of leukemogenesis.

Aryl hydrocarbon receptor-kynurenine axis promotes oncogenic activity in BCP-ALL.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the most common childhood cancer, originates from lymphoid precursor cells in bone marrow committed to the B-cell lineage. Environmental factors and genetic abnormalities disturb the normal maturation of these precursor cells, promoting the formation of leukemia cells and suppressing normal hematopoiesis. The underlying mechanisms of progression are unclear, but BCP-ALL incidence seems to be increasing in parallel with the adoption of modern lifestyles. This study hypothesized that air pollution and haze are risk factors for BCP-ALL progression. The current study revealed that indeno(1,2,3-cd)pyrene (IP), a major component of polycyclic aromatic hydrocarbons (PAHs) in air, promotes oncogenic activities (proliferation, transformation, and disease relapse) in vitro and in vivo. Mechanistically, IP treatment activated the aryl hydrocarbon receptor (AHR)-indoleamine-2,3-dioxygenase (IDOs) axis, thereby enhancing tryptophan metabolism and kynurenine (KYN) level and consequent promoting the KYN-AHR feedback loop. IP treatment decreased the time to disease relapse and increased the BCP-ALL cell count in an orthotopic xenograft mouse model. Additionally, in 50 clinical BCP-ALL samples, AHR and IDO were co-expressed in a disease-specific manner at mRNA and protein levels, while their mRNA levels showed a significant correlation with disease-free survival duration. These results indicated that PAH/IP exposure promotes BCP-ALL disease progression.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Genetic Profiling of Pediatric Patients with B-Cell Precursor Acute Lymphoblastic Leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous leukemia subgroup. It has multiple sub-types that are likely to be classified by prognostic factors. Following a systematic literature review, this study analyzed the genes correlated with BCP-ALL prognosis ( IKZF1, PAX5, EBF1, CREBBP, CRLF2, JAK2, ERG, CXCR4, ZAP70, VLA4, NF1, NR3C1, RB1, TSLP, ZNRF1, and FOXO3A) , specifically their nucleotide variations and expression profiles in pediatric BCP-ALL samples. The study included 45 pediatric BCP-ALL patients with no cytogenetic anomaly and a control group of 10 children. The selected genes' hot-spot regions were sequenced using next-generation sequencing, while Polymorphism Phenotyping v2 and Supplemental Nutrition Assistance Program were used to identify pathogenic mutations. The expression analysis was performed using quantitative real-time polymerase chain reaction. The mutation analysis detected 328 variants (28 insertions, 47 indels, 74 nucleotide variants, 75 duplications, and 104 deletions). The most and least frequently mutated genes were IKZF1 and CREBBP , respectively. There were statistically significant differences between patients and controls for mutation distribution in eight genes ( ERG, CRLF2, CREBBP, TSLP, JAK2, ZAP70, FOXO3A, and NR3C1 ). The expression analysis revealed that JAK and ERG were significantly overexpressed in patients compared with controls (respectively, p = 0.004 and p = 0.003). This study combined genes and pathways previously analyzed in pediatric BCP-ALL into one dataset for a comprehensive analysis from the same samples to unravel candidate prognostic biomarkers. Novel mutations were identified in all of the studied genes.

Toll-like Receptor-3 Ligation Induces Pro-Survival Signaling in Pediatric Acute Lymphoblastic Leukemia

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common cancer in children, with a peak age of incidence of 2-5 years. In the majority of pediatric cases, B-ALL is initiated in utero, with full transformation from pre-leukemia to leukemia being driven by additional oncogenic events acquired in early childhood. Surprisingly, common in utero genetic events lead to the expansion of pre-leukemic clones in >1% of all newborns, yet only 1 in 100 of these infants go on to develop clinically overt leukemia in their lifetime. This suggests the presence of certain protective and/or non-protective mechanisms that influence the fate of pre-leukemic clones.While several epidemiological studies of early-life infection indicate a consequent increased risk for ALL, protective effects of infection exposure against ALL have also been reported. An infectious agent responsible for pro- or anti-leukemic effects has not been identified and a mechanistic explanation for the seemingly contradictory effects of early-life infection has not been defined. We have previously reported the ability of immune stimulation with infection-associated toll-like receptor (TLR) agonists to reduce pre-leukemic cell burden and delay leukemia onset in the Eμ-RET transgenic mouse model of hyperdiploid B-ALL. We observed that while TLR9 (CpG) and TLR7/8 (R848) ligands were effective at reducing viable pre-leukemic and leukemic cells, a TLR3 ligand (PolyI:C) appeared to have an opposite effect. In this study, we further investigate the potential pro-leukemic effect of TLR3 ligands (pathogenic dsRNA) on abnormal BCP cell behavior.Purified pre-leukemic or leukemic cells from Eu-ret mice were treated in vitro with TLR agonists and cell viability was assessed after 48 hours. In contrast to the reduced viability observed after exposure to TLR7/8 (R848) and TLR9 (CpG) ligands, a ~1.5-fold increase in viable cell recovery, compared to untreated cells, was observed after treatment with PolyI:C (p <0.05). Since PolyI:C binds other receptors in addition to TLR3, we then assessed PolyA:U, a synthetic dsRNA molecule signaling exclusively through TLR3. Treatment of purified pre-leukemic and leukemic cells with PolyA:U lead to an increase in viable cell recovery compared to untreated cells (p < 0.01), implicating TLR3 as the relevant receptor. This viable cell recovery correlated with reduced apoptosis and an increased proliferation index in PolyA:U treated samples.To assess the relevance of this TLR-induced activity for clinical B-ALL, we evaluated the responses of patient-derived B-ALL samples. A similar survival benefit was observed with primary B-ALL samples treated with TLR3, but not other TLR ligands. Consistent with observations from primary murine leukemia, CpG and R848 were able to induce in vitro killing of human leukemia cells, while PolyA:U promoted increased leukemia cell viability.As the overall outcome of early-life infection will also involve TLR-induce immune responses, we next assessed the effect of TLR3 agonists in the presence of immune effector cells. Bulk splenocytes from 1- to 12-week-old Eu-RET mice were cultured in the presence or absence of TLR agonists and viable cell recovery after 48 hours measured. In the presence of immune effector cells, the impact of pro-survival TLR3 signalling was diminished in an age-dependent manner, implicating neonatal immunity in the abnormal pro-leukemic response induced by TLR3 ligation.Overall, our results reveal previously unrecognized pro-survival signalling through TLR3 that could contribute to an early-life infection-driven progression of B-ALL. We are currently exploring the signaling pathway(s) responsible for this effect and evaluating the immune variables that influence pre-leukemia cell fate in vivo. This study will significantly enhance our mechanistic understanding of immune-mediated modification of B-ALL progression induced by early-life infection exposure. DisclosuresNo relevant conflicts of interest to declare.

IL7R Targeting Using OSE-127 Shows Robust Anti-Leukemic Activity in B-Cell Precursor Acute Lymphoblastic Leukemia Xenografts

Despite the favorable prognosis of B cell precursor acute lymphoblastic leukemia (BCP-ALL), relapse remains a clinical challenge. Antibody-based immunotherapies like Blinatumomab have improved BCP-ALL outcome, yet these can quickly turn ineffective due to CD19 antigen escape. Therefore, novel targeted immunotherapy options are urgently needed.We previously showed that high IL7R expression associates with adverse outcome in BCP-ALL patients and that IL7R targeting using laboratory-grade blocking antibodies significantly reduced leukemic burden in E2A-PBX1 and BCR-ABL patient derived xenografts (PDX) (Alsadeq et al., Blood, 2017; Abdelrasoul et al., Nat. Commun., 2020). Here we used the humanized monoclonal antibody OSE-127, a full IL7R antagonist which is not internalized by target cells and prevents IL7R heterodimerization and subsequent downstream signaling (Belarif et al., Nat. Commun. 2018, Belarif et al. JCI, 2019). Importantly, OSE-127 has recently been positively evaluated in a phase 1 study conducted in 63 healthy volunteers receiving either a single dose or two doses two weeks apart (EUDRACT 2018-001832-22). OSE-127 demonstrated an excellent safety and tolerability profile showing no signs of lymphopenia, cytokine release syndrome or T-cell compartment alterations. Based on these preclinical and clinical results, we investigated the anti-leukemic efficacy of OSE-127 immunotherapy in PDX models of BCP-ALL.First, to identify patients that may benefit from IL7R-immunotherapy, we measured IL7R surface expression in diagnostic BCP-ALL patient samples of different cytogenetic subgroups via flow cytometry.We detected IL7R-positivity (defined as ≥10% IL7R + BCP-ALL cells) in 52% (49/94) of patient samples including BCR-ABL + (25%; 5/20), TEL-AML1 + (41%; 7/17) and B-other (40%, 8/20) samples. Particularly high numbers of IL7R + patients were found in the E2A-PBX1 + and MLL-rearranged (MLLr) BCP-ALL subgroups (79%; 19/24 and 85%; 11/13 IL7R + patient samples, respectively).Next, we tested the efficacy of OSE-127 in NSG mice injected with PDX cells from two different E2A-PBX1 + patients (63.0% and 89.6% IL7R-positive blasts, respectively) modelling an MRD-situation by starting treatment one day after injection of PDX cells. While 100% of control animals developed overt leukemia, 0% of the OSE-127-treated animals developed the disease, with 10/10 and 7/10 mice per PDX-group being MRD-negative by the time of sacrifice. Of note, OSE-127-therapy was also effective in corresponding PDX models when therapy was initiated upon detection of 1% BCP-ALL cells in the peripheral blood, modelling an overt leukemia situation (P<0.0001 in both cases).To investigate the efficacy of OSE-127 in a variety of BCP-ALL samples, we conducted a preclinical phase II-like study using PDX from different cytogenetic patient subgroups (8E2A-PBX1 +, including 1 matched diagnostic/relapse sample, 1 BCR-ABL, 1 MLLr and 1 ETV6-NTRK3). Two mice per PDX were injected, randomly assigned to control or OSE-127 treatment groups and therapy was initiated when >1% blasts were detected in the peripheral blood. Response to OSE-127 was detected in 9/11 (82%) PDX samples in vivo, including BCR-ABL +, MLLr and E2A-PBX1 +-relapse samples. Accordingly, OSE-127 immunotherapy led to a significant increase in median overall survival (85 days versus 55 days; P=0.0122). Of note, OSE-127 therapy response associated with IL7R expression levels on BCP-ALL cells and no significant downregulation of the IL7R antigen was observed upon OSE-127 treatment.Taken together, our data demonstrate that IL7R-targeting using OSE-127, which has already demonstrated a good safety profile in healthy volunteers, is an efficient approach for BCP-ALL immunotherapy and may be particularly beneficial for patients with high IL7R-expression and/or relapsed/refractory disease after CD19-directed therapy. DisclosuresLenk: OSE Immunotherapeutics: Research Funding. Baccelli: OSE Immunotherapeutics: Current Employment, Other: Author of patents related to anti-IL7R antibodies. Corallo: OSE Immunotherapeutics: Current Employment. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; Novartis: Honoraria, Other: research support; SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Poirier: OSE Immunotherapeutics: Current Employment, Other: Author of patents related to anti-IL7R antibodies. Schewe: OSE Immunotherapeutics: Research Funding; Bayer: Other: Advisory Board; SOBI: Other: Advisory Board; Jazz Pharmaceuticals: Other: Advisory Board.

Abstract 3033: Molecular classification of pediatric high-risk leukemias using expression profiles of multimodally expressed genes

Abstract Introduction: Leukemia is the most common cancer in children, accounting for approximately one third of all malignancies that occur in the pediatric age group. Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML) account for most leukemia diagnosed in this age group. While known markers for poor prognosis include higher age, higher white blood cell count at diagnosis and certain translocations, innovative approaches in tumor RNA sequencing (RNA-Seq) data analysis can discover novel prognostic factors that could be exploited for future therapeutic development in fusion-negative ALL and AML. Methods: To reveal gene expression signatures among fusion-negative leukemias, we used a novel unsupervised analysis model called Hydra. Hydra uses a Dirichlet process mixture model to detect multimodally expressed genes to use in characterizing clusters within cancer cohorts. This approach can detect subtle yet robust differences in gene expression without the reliance on reference normal RNA-Seq datasets. The Hydra model reveals clusters of the cancer cohort, and differences among these clusters can be investigated by finding enriched pathways via Gene Set Enrichment Analysis (GSEA). The cluster-specific enriched pathways can be used in conjunction with survival data to determine how certain pathways are associated with outcome. This analysis used publicly available data from the National Cancer Institute (NCI) Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database that was uniformly processed by the Treehouse Childhood Cancer Initiative. Results: First, 202 fusion-negative AML and fusion-negative B-cell precursor ALL samples were run through Hydra and five clusters were identified. These clusters had different enriched pathways, such as high mitochondrial activity, high cell proliferation, and high cell signaling. Though these are characteristics of all cancer cells, each cluster demonstrated that one pathway was most distinctive of those samples. Most clusters were differentiated by disease, however, one cluster with enriched heme metabolism and immunoglobulin pathways contained almost equal amounts of AML and ALL samples, suggesting that specific cohorts of AML and ALL patients had increased inflammatory response. Another cluster contained 72 AML samples and 4 ALL samples. The four ALL samples in this cluster showed lowered expression of CD19, a B-cell lineage immune marker, and elevated expression of CD14, a myeloid lineage immune marker. These ALL patients exhibited genomic characteristics of AML, which may suggest a more specialized treatment regimen. Discussion: Despite extensive characterization of pediatric high-risk leukemias using genomic approaches, there is ample opportunity to study RNA-Seq-derived gene expression profiles to help accurately diagnose and treat pediatric patients. Citation Format: Sneha S. Jariwala, Alfred Geoffrey Lyle, Jacob Pfeil, Lauren Sanders, Holly C. Beale, Ellen T. Kephart, Katrina Learned, Allison Cheney, Olena M. Vaske. Molecular classification of pediatric high-risk leukemias using expression profiles of multimodally expressed genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3033.

Integrated Genetic/Epigenetic Analysis Revealed High Heterogeneity of Acute Lymphoblastic Leukemia in Down Syndrome

BackgroundChildren with Down syndrome (DS), which caused by an extra copy of chromosome 21, have a 20-fold increased risk for acute lymphoblastic leukemia (ALL) compared with non-DS children. Recent genome-wide studies have revealed that DS-ALL have uncommon genetic alterations such as mutations in JAK2 , NRAS , and KRAS , mutations or overexpression of CRLF2 , suggesting the unique biological features of DS-ALL distinct from non-DS ALL. However, most of previous studies regarding molecular analysis of DS-ALL have been limited to genomic and transcriptomic characterizations, and no study showed comprehensive genetic/epigenetic overview in DS-ALL.Materials and MethodsOur cohort included 179 pediatric B-cell precursor ALL samples (61 DS-ALL and 118 non-DS-ALL). Biological subtypes are detailed in Table 1. We performed targeted deep sequencing for seven ALL-related genes in 61 DS-ALL, SNP array analysis in 43 DS-ALL, and whole transcriptome sequencing (WTS) in 25 DS-ALL and 118 non-DS-ALL samples. WTS data from 153 ALL in TARGET database were also used in this study as a validation cohort. Additionally, methylation array analysis was performed in 36 DS-ALL, 17 non-DS-ALL, and 20 DS patients without ALL. To validate gene mutations detected by WTS, Sanger sequencing was also conducted. To cluster gene expression profiling, we applied the hierarchical clustering method for expression profiling and consensus clustering for methylation profiling.ResultsTargeted deep sequencing revealed frequent gene mutations of JAK2 (23%) and RAS (31%) pathway. SNP array analysis elucidated several recurrent chromosomal deletions affecting actionable gene loci; IKZF1 (33%), PAX5 (30%), EBF1 (15%), CDKN2A (34%), and RB1 (7%). Gains of whole chromosome X (18%) and 8 (10%) were also recurrently identified. Expression profiling stratified 143 samples into 6 clusters (E1-E6 clusters), which were well correlated with biological subtypes of ALL (Figure 1). Intriguingly, DS-ALL cases were clustered into four clusters, suggesting the heterogeneity of DS-ALL. Cluster E3 included one DS-ALL, which was clustered to the branch of PAX5 -rearranged cases. SNP array analysis revealed this case had focal amplification of PAX5 . All DS-ALL cases with ETV6 - RUNX1 fusion were classified into cluster E4, which also included all ETV6 - RUNX1 fusion cases of non-DS-ALL. This cluster included one DS-ALL case without ETV6 - RUNX1 fusion. This particular case had a homozygous deletion of ETV6, implicating ETV6 - RUNX1 -like signature. DS-ALL with high hyperdiploidy (HeH) were classified into cluster E5, which also included most of HeH samples of non-DS-ALL. Among other 5 DS-ALL cases in this cluster, 2 cases showed IGH - CEBPA fusion and IGH - CEBPD fusion, respectively. Cluster E6 was characterized by BCR - ABL1 fusion and Ph-like expression profiles. In our cohort, we identified 7 DS-ALL cases as Ph-like signature, which included all JAK2 mutated samples. In this cluster, PAX5 mutation and IKZF1 mutation were detected in 2 DS-ALL cases, respectively. Notably, by using 250 genes in our hierarchical clustering, TARGET database samples were divided to similar 6 clusters (Figure 2). On the other hand, Methylation array analysis revealed unique hypermethylation of promoter regions of RUNX1 in DS-ALL. Methylation of this regions were confirmed in B-cell of DS without ALL, but not in non-DS-ALL. Furthermore, methylation array analysis categorized DS-ALL into 3 clusters (Figure 3). Cluster M1 included IGH - CEBPA fusion and IGH - CEBPD fusion cases. This cluster had worse prognosis than other 2 clusters.ConclusionOur integrated analysis enlightened the genetic and epigenetic heterogeneity of DS-ALL. Importantly, DS-ALL had genetic features corresponding to each biological subtypes same as non-DS-ALL. Our analyses also elucidated that DS-ALL had unique subtypes such as IGH - CEBPA fusion, IGH - CEBPD fusion, or PAX5 alterations. It may be a reason for worse prognoses of DS-ALL. Our result indicated risk classification or therapy of DS-ALL should be tailored by each biological subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High PDGFRA Expression Does Not Serve As Effective Therapeutic Target in ERG-Deleted B-Cell Precursor Acute Lymphoblastic Leukemia

Background and aimsSeveral oncogenic aberrations of receptor tyrosine kinases occur in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we describe a subgroup of pediatric patients lacking common chromosomal aberrations but with high gene expression of platelet-derived growth factor receptor alpha (PDGFRA). Oncogenic fusions involving the tyrosine kinase domain of PDGFRA are recurrent in eosinophilic leukemia and are responsive to kinase inhibitors (Cools et al. N Engl J Med 2003). One case with a FIP1L1-PDGFRA fusion was reported in adult BCP-ALL (Roberts et al. J Clin Oncol 2016). Therefore, we tested for genomic aberrations underlying the overexpression of PDGFRA and evaluated the inhibition of PDGFRα as treatment option in pediatric BCP-ALL.MethodsExpression profiling was performed using Affymetrix U133 plus 2.0 arrays. BCR-ABL1 -like cases were identified using the 110 probe set expression signature (Den Boer et al. Lancet Oncol 2009). High PDGFRA was defined as log2 expression of probe set 203131_at >5.9. FISH was performed using Cytocell FIP1L1/CHIC2/PDGFRA probes. Targeted locus amplification (de Vree et al. Nat Biotechnol 2014) was performed using custom-designed primers. Cells were treated for 10 minutes with 10 ng/mL PDGF-BB with or without 10 µM imatinib or 1 µM CP673451 and analyzed by western blot. Cell viability after 4 day exposure to imatinib (0.2µM - 1.6µM - 12.5µM) or CP673451 (0.02µM - 0.16µM - 1.25µM) was assessed by flow cytometry.ResultsGene expression analysis of 654 pediatric ALL cases identified high expression of PDGFRA in 26 of 574 BCP-ALL cases (4.5%). High PDGFRA expression was exclusively found in cases belonging to the B-other (n=22) or BCR-ABL1 -like (n=4) subtypes, and not present in ETV6-RUNX1, high hyperdiploid, TCF3-PBX1, BCR-ABL1 and KMT2A -rearranged BCP-ALL subtypes or T-ALL. PDGFRA expression was confirmed by RT-qPCR, and also found in the BCP-ALL cell line Nalm6. Using RT-PCR we did not detect any of the six reported PDGFRA fusions, which involve FIP1L1, STRN, BCR, CDK5RAP2, ETV6, and KIF5B, nor did we detect novel structural aberrations affecting PDGFRA by FISH. We studied the PDGFRA locus at high resolution by targeted locus amplification. Following DNA cross-linking, digestion, and proximity-based re-ligation, targeted amplification was performed from viewpoints in intron 2 and exon 15 of PDGFRA. In the eosinophilic cell line EOL1 we confirmed an interstitial deletion on chromosome 4 which rearranges FIP1L1 intron 12 to PDGFRA exon 12. The results obtained from Nalm6 and primary BCP-ALL samples with high PDGFRA expression did not indicate genomic aberrations of PDGFRA.ERG deletions were present in 10 of 16 PDGFRA -high cases (63%), compared with 0% in PDGFRA -low BCP-ALL cases (n=71; p<0.001). Vice versa, all ERG-deleted cases expressed high levels of PDGFRA . Recently, a novel BCP-ALL subtype was described with rearrangements of the transcription factor DUX4 and deletion of ERG (Lilljebjörn et al. Nat Commun 2016; Yasuda et al. Nat Genet 2016; Zhang et al. Nat Genet 2016). Furthermore, the high PDGFRA-expressing cell line Nalm6 carries a DUX4 rearrangement (Yasuda et al. Nat Genet 2016) and PDGFRA is highly expressed in DUX4 BCP-ALL and its transcription start site directly bound by DUX4 (Zhang et al. Nat Genet 2016). Therefore, we regard it as highly likely that the PDGFRA-high cases represent this novel DUX4- rearranged subtype.Because transcription factors are difficult to target with small molecule inhibitors, PDGFRα inhibition could represent a treatment option. Exposure to the PDGFR inhibitors imatinib and CP673451 reduced phosphorylation of PDGFRα in Nalm6 and primary BCP-ALL cells. However, ex vivo drug exposure revealed no cytotoxic effect of the PDGFR inhibitors on PDGFRA-high BCP-ALL cells, either in the presence or absence of the ligand PDGF-BB. Interestingly, we found a marked ex vivo sensitivity towards prednisolone in PDGFRA-high cases, in line with the good prognosis reported for ERG -deleted/DUX4-rearranged BCP-ALL.ConclusionsWe found high PDGFRA expression, in the absence of genomic aberrations of the PDGFRA locus, in a subgroup of BCP-ALL patients with frequent ERG deletions. PDGFR inhibition caused abrogation of PDGFRα phosphorylation but was not cytotoxic in PDGFRA-high expressing BCP-ALL cells. Therefore, high PDGFRA expression does not serve as effective therapeutic target in ERG -deleted BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Transcriptome Analysis Identifies Ligand-Receptor Pairs Associated With BCP-ALL Prognosis.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a blood cancer that originates from the abnormal proliferation of B-lymphoid progenitors. Cell population components and cell–cell interaction in the bone marrow microenvironment are significant factors for progression, relapse, and therapy resistance of BCP-ALL. In this study, we identified specifically expressed genes in B cells and myeloid cells by analyzing single-cell RNA sequencing data for seven BCP-ALL samples and four healthy samples obtained from a public database. Integrating 1356 bulk RNA sequencing samples from a public database and our previous study, we found a total of 57 significant ligand–receptor pairs (24 upregulated and 33 downregulated) in the autocrine crosstalk network of B cells. Via assessment of the communication between B cells and myeloid cells, another 29 ligand–receptor pairs were discovered, some of which notably affected survival outcomes. A score-based model was constructed with least absolute shrinkage and selection operator (LASSO) using these ligand–receptor pairs. Patients with higher scores had poorer prognoses. This model can be applied to create predictions for both pediatric and adult BCP-ALL patients.

Pre-Clinical Efficacy of the Novel Kinase Inhibitor Nintedanib on PAX5 Fusion Genes in Pediatric Ph-like B-Cell Precursor Acute Lymphoblastic Leukemia

Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120. This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120. We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement. Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells. In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent. Disclosures No relevant conflicts of interest to declare.

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53’s wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo. In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo. Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.