Targeting NOTCH1 in combination with antimetabolite drugs prolongs life span in relapsed pediatric and adult T-acute lymphoblastic leukemia xenografts

Thanks to the current therapeutic protocols, children and adults’ affected by T-cell acute lymphoblastic leukemia (T-ALL) [1] present an overall survival (OS) rate that reaches 85–90% and 40–50%, respectively [2, 3]. Nevertheless, around 25–40% of pediatric and adult T-ALL patients still experience relapses, with an OS around 25% for both patient groups [4, 5]. Furthermore, for relapsed T-ALL patients, except from Hematopoietic Stem Cell Transplantation and the intensification of the therapeutic regimen administered after the first diagnosis, no novel therapeutic options are available so far [1, 4, 5]. Therefore, the identification of novel therapeutic approaches are necessary to treat T-ALL relapsed patients thus preventing a poor outcome. In this light, TP53 mutations and deletions have been shown to occur more frequently at relapse and are adversely associated with second-line therapy survival [6]. Additionally, 60% of T-ALL patients present activating NOTCH1 mutations or alterations in its ubiquitin ligase FBXW7 [7, 8], suggesting NOTCH1 signaling pathway as a possible therapeutic target to overcome relapsed T-ALL. In this regard, several preclinical studies have been reported either directly or indirectly inhibiting NOTCH1 signaling [9‐11], but few reports on relapsed T-ALL treatment have been published so far [12]. Taking advantage of our previous studies [9] here we aimed to assess if NOTCH1 signaling inhibition by the specific monoclonal anti-NOTCH1 antibody (OMP-52M51) would be effective at relapse, exploiting NSG mice xenograft models established from both pediatric (PDTALL46, PDTALL39 and PDTALL47) and adult (PDTALL-AD2R and PDTALL-AD4) relapsed NOTCH1 and TP53 mutated T-ALL samples (Additional file 1: Table S1 and Fig. S1A-B).

As first, we treated PDX mouse models with anti-NOTCH1 monotherapy, started 2 days after i.v. injection of T-ALL relapse cells into mice, and we observed a clear leukemia burden reduction in the peripheral blood (PB) (Fig. 1A-C and Additional file 1: Fig. S2A upper panel), bone marrow (BM) and spleen (Fig. 1A-C and Additional file 1:  Fig. S2A bottom panel) in 4 out of 5 T-ALL PDXs. Only PDTALL-AD2R was apparently not responding to treatment (Additional file 1: Fig. S2B), probably due to the almost undetectable expression of NOTCH1 target genes (Additional file 1: Fig. S1) suggesting the absence of a NOTCH1 pro-survival signaling dependence, despite the presence of a NOTCH1 PEST domain mutation. RNAseq analysis from in vivo PDTALL46 cells treated or not with OMP-52M51 unveiled that the anti-NOTCH1 therapy causes a significant down regulation of NOTCH1 signaling, histidine and tyrosine metabolism as well as purine metabolism which can be targeted by FDA-approved antimetabolites drugs used in T-ALL treatment (Fig. 1D, Additional file 1: Fig. S3 and Table S2). Accordingly, the in vitro apoptosis Caspase 3/7 assay on PDTALL46 and PDATALL39 primary T-ALL cells revealed the most significant IC₅₀ index decrease in the combination between anti-NOTCH1 and antimetabolites used during the consolidation/maintenance phases [Cytarabine (AraC), methotrexate (MTX) and 6-mercaptopurine (6MP)], (Fig. 1F, Additional file 1:  Fig. S4 and Table S3), compared to drugs administered along the induction phase therapy [vincristine (Vinc) and daunorubicin (Dauno)] (Fig. 1E). Thus, starting from these results and based on the kinetics of PDTALL46 leukemia growth (Additional file 1: Fig. S5A), we started the in vivo treatment (day 11) with the anti-NOTCH1 alone or in combination with COMBO1 (Vinc, Dauno, Dexa) or COMBO2 (AraC, MTX, 6MP) schedule when the percentage of CD5⁺/CD7⁺ circulating blasts in the PB of PDTALL46 was around 1–2% (Additional file 1: Table S4, Fig. 1G). Interestingly, we observed a significant reduction of CD5⁺/CD7⁺ blasts in mice treated with the anti-NOTCH1 antibody in combination with both therapeutic schedules (COMBO1/2) in all the compartments (PB, BM and spleen) as well as a decrease in spleen weight (Additional file 1: Fig. S5B) compared to controls or single arm treatments (Fig. 2A-B). Importantly, mice treated with both the anti-NOTCH1 antibody and COMBO2 showed the best therapeutic effect (Fig. 2B). These results were further confirmed in the pediatric PDTALL39 (Fig. 2C and Additional file 1: Fig. S6A) and in the adult PDTALL-AD4 (Fig. 2D and Additional file 1: Fig. S6B) PDX models, although in the latter with less efficacy when compared to the pediatric one, probably due to the fact that adult T-ALL have lower response rate to chemotherapy and thus result more difficult to treat.

Finally, we performed survival experiments by administrating the anti-NOTCH1 and COMBO2 treatments alone or in combination, and stopped the treatments at 20-40% of circulating blasts in control mice PB (Additional file 1: Fig. S7A-C). In agreement with the efficacy in vivo studies, all PDX mice models treated with the anti-NOTCH1 and COMBO2 therapy showed a significantly (p < 0.001) longer life span survival, between 20 and 290 days, compared to the COMBO2 alone treated group (0-100 days) (Fig. 2E-G), thus corroborating the hypothesis that NOTCH1 targeted therapy improves therapeutic efficacy of antimetabolite drugs (Fig. 2H).

In conclusion, altogether these results provide a rationale for a novel therapeutic strategy that provides NOTCH1 inhibition in combination with antimetabolites drugs in T-ALL relapsed pediatric and adult patients, for whom so far no other therapeutic options are available.