PAX5 fusion genes are frequent in poor risk childhood acute lymphoblastic leukaemia and can be targeted with BIBF1120

Abstract

Background: Despite intensive risk-based treatment protocols, 15% of paediatric patients with B-Cell Precursor Acute Lymphoblastic Leukaemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups, like PAX5 translocated.

Methods: We considered 289 childhood BCP-ALL cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols and we performed extensive molecular profiling, integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS. We developed preclinical strategies to target PAX5 fusion genes.

Findings: We identified 135 cases without recurrent genetic rearrangements. Among them, 59 patients (43·7%) had a Ph-like signature; the remaining cases were identified as ERG-related (26%), High-Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%). A poor prognosis was associated with the Ph-like signature, independently from other high-risk features. Interestingly, PAX5 was altered in 54·4% of Ph-like compared to 16·2% of non-Ph-like cases, with 7 patients carrying PAX5 fusions (PAX5t), involving either novel (ALDH18A1, IKZF1, CDH13) or known (FBRSL1, AUTS2, DACH2) partner genes. PAX5t cases have a specific driver activity signature, extending to multiple pathways including LCK hyperactivation. Among FDA-approved drugs and inhibitors, we selected Dasatinib, Bosutinib and Foretinib, in addition to Nintedanib, known to be LCK ligands. We demonstrated the efficacy of the LCK-inhibitor BIBF1120/Nintedanib, as single agent or in combination with conventional chemotherapy, both ex vivo and in patient-derived xenograft model, showing a synergistic effect with dexamethasone.

Interpretation: This study provides new insights in high-risk Ph-like leukaemia and identifies a potential therapy for targeting PAX5-fusion poor risk group.

Funding: Ricerca Finalizzata-Giovani Ricercatori (Italian Ministry of Health), AIRC, Transcall, Fondazione Cariparo.

Keywords: BIBF1120; Childhood ALL; Nintedanib; PAX5 fusion genes; Ph-like ALL.

Figure 1

(a) Two dimensional tSNE plot of 289 paediatric BCP-ALL cases based on the top 1000 variable genes of gene expression data. The BCP-ALL subgroups are represented with different colours. Colour label indicated the different subgroups. Yellow highlights a subgroup of Ph-like patient carrying PAX5-translocations (PAX5t). Groups recognized as “like” cluster together with samples carrying the associated translocations, which is attributed to a similar gene expression profile. (b) Plot summarizing the molecular characterization of B-other cohort in childhood ALL: assessment of specific genetic subtypes, as reported in details in Supplementary Table S2.

Figure 2

Outcome analysis of AIEOP B-Other cohort, in terms of EFS (on the left column) and CIR (in the middle column) and OS (in the right), comparing Ph-like vs. non-Ph-like patients overall (a), in no High Risk (b), in HR patients (c), respectively; (d) EFS, CIR and OS analyses according to the fusion classes reveal statistical significance in Ph-like cases, classified accordingly to different class fusions, such as ABL/JAK-class, PAX5t, other fusions and negative after NGS analysis.

Figure 3

PAX5 translocated cases (or PAX5t) have a specific interactome signature. (a) Schema of NetBID2 analysis in BCP-ALL at diagnosis to identify driver in PAX5t BCP-ALL patients. To construct the BCP-ALL interactome the SJARCNe algorithm was employed on gene expression profiles of BCP-ALL patients (n = 289), and driver gene networks in PAX5t patients were identified in comparison with Ph-like or all other BCP-ALL subtypes. (b) Heatmap of the top 44 predicted driver's activity (DA) and (c) differentially expressed (DE) in the BCP-ALL interactome based on PAX5t patients. Drivers were ranked according to p-value. Colour code was generated by z-score. (left column= PAX5t vs. other BCP-ALL subtypes; middle column=PAX5t vs. Ph-like, right column=Ph-like vs. other BCP-ALL subtypes). (d) Boxplot of LCK activity by NetBID2 analysis of PAX5 translocated patients compared between Ph-like and all other BCP-ALL subtypes using gene expression data. p-value was calculated using Welch T-test. (e) Boxplot of LCK activity in BCP-ALL patients with known PAX5 status (N = 131). p-value was calculated by Welch t-test. PAX5amp: patients with amplification of PAX5 gene; PAX5del: patients with deletion of PAX5 gene; PAX5t: patients with translocation of PAX5 gene; PAX5wt: patients with PAX5 gene without aberrations. (f) Comparative cellular viability of different sub-groups of leukemia (PAX5t, MLLr or CRLF2r) samples and control-B-lymphoblastoid cell lines (CTR-B-LCSs), measured by ATP-Glo based luminescent based assay after exposure of the depicted drugs, whereas Staurospsorin was taken as a positive control. Drug sensitivity scores (DSS) are plotted as a clustered heat map, followed by unsupervised hierarchical clustering. The horizontal and the vertical axis of the dendrogram illustrates the dissimilarity between clusters, whereas the color of the cell is related to its position along with a DSS gradient. The p-values are calculated with the one-way Anova test related to PAX5t group, **** (p < 0·0001), *** (p < 0·001), ** (p < 0·01); * (p < 0·05); ns = not significant.

Figure 4

Ex vivo treatment by BIBF1120 either alone or in combination with Dexamethasone (a), Vincristine (b) or Asparaginase (c). Experiments have been performed in presence of different PAX5 fusion genes, such as PAX5::AUTS2 in Pt. n°1 and n°2, or alternatively carrying PAX5::DACH2 as in Pt. n°3 or PAX5::SOX5 in Pt. n°4. After drug treatment, viability has been determined, as difference from apoptosis and necrosis, by Annexin V-PE and 7-AAD staining. Untreated samples (ut) have been analysed as internal control.

Figure 5

In vivo treatment by BIBF1120 either administered alone or in combination with Dexamethasone. The experimental plan is depicted in panel (a): NSG mice were transplanted with human leukemic cell carrying different PAX5 fusion genes, namely PAX5::AUTS2 as in Pt. n°1 (panels b-g) or PAX5::DACH2 as in Pt. n°3 (panels h-o). Human leukaemia engraftment was evaluated in hematopoietic tissues prior to start treatment in Bone Marrow and Peripheral Blood (panels b and h, in both patients, respectively) and after two weeks of treatment, in all hematopoietic organs, such as Bone Marrow for Pt.1 (panel c) and Pt.3 (panel i), in Spleen as percentage (panel d and l) and weight (panels e and m), in Peripheral Blood, f and n) and in Central Nervous System Meninges (g and o). Data are expressed as percentages of human CD10⁺ cells. Statistical analyses have been performed by GraphPad Prism software and T test is shown as *p < 0·05, **p < 0·01, ***p < 0·001.

Figure 6

LCK and Akt pathway involvement in leukemic cells with PAX5 fusion genes. (a) A schematic view of molecular hypothesised mechanism of LCK and Akt interaction. After in vivo BIBF1120 treatment vs. vehicle mice, in (b) pt.#1 PAX5::AUTS2 and (c) pt.#3 PAX5::DACH2, respectively. PDPK1, pAKT-Ser473 and -Thr308, pS6 and 4pEBP1 are analysed by FACS. Data are expressed as ratio of Phosphorylation levels, considering vehicle phosphoprotein levels equal to 1 (dotted line) and symbols are correspondent to single BIBF1120-treated mice. Statistical analyses have been performed by GraphPad Prism software and T test is shown as *p < 0·05, **p < 0·01, ***p < 0·001.