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V. Agnusdei et al.
   PDTALL8 (Figure 1B). Notably, the dynamic tracking of the percentage of T-ALL cells in blood uncovered substan- tially different profiles in these models. In fact, we observed a steep increase in the case of PDTALL8 model, compared with the mild increase observed in the PDTALL19 model (Online Supplementary Figure S2), sug- gesting different mechanisms of adaptation to OMP52M51.
Following treatment of T-ALL PDX with OMP52M51, we previously observed a strong reduction of Notch target gene expression in good responders and we considered this phenomenon as indirect evidence of reduced Notch signaling.9 Therefore, we wondered if this inhibition was still present in mice which developed resistance to OMP52M51 therapy. To investigate this hypothesis, we analyzed expression levels of 21 Notch target genes by qPCR. In the PDTALL8 model, expression levels of Notch target genes were very similar in OMP52M51-resistant and control cells (Figure 1C, top). On the contrary, in PDTALL11 xenograft several genes were significantly downregulated in cells recovered from OMP52M51-resis- tant compared to control mice (Figure 1C, middle). In the PDTALL19 model, inhibition of Notch signaling was maintained in resistant cells (Figure 1C, bottom) and it was comparable to that previously measured following acute treatment with OMP52M51.9
Serial transplantation experiments disclosed two types of resistance to OMP52M51
To elucidate whether resistance to OMP52M51 was a stable trait, we performed serial transplantation experi- ments. T-ALL cells from the spleen of donor mice resist- ant to OMP52M51 were i.v. injected into naïve recipient mice, which were then weekly treated with OMP52M51 or control antibody according to the standard protocol until the appearance of the signs of disease (Figure 2A). We found that resistance was a stable trait in the PDTALL8 model, the PDX in which resistance developed 156 days after OMP52M51 treatment (late onset). In fact, in the serial transplantation experiment, both OMP52M51-treated and control mice developed leukemia 39 days after T-ALL cell injection (Figure 2B). In contrast, resistance to OMP52M51 was lost upon serial transplantation in PDTALL19 model, the PDX with early onset of resistance. PDTALL19 cells recovered from the spleen of OMP52M51-resistant mice and injected into näїve mice were initially sensitive to OMP52M51 treat- ment, as no evidence of T-ALL cells was found in the blood of these mice at day 18, when control mice were sacrificed. When administration of OMP52M51 was repeated, however, mice became gradually resistant to therapy and they were eventually sacrificed 41 days after T-ALL cell injection, a time point very similar to that observed in the initial experiment (Figure 2B). It is impor- tant to note, however, that following repeated (n=5) cycles of treatment of PDTALL19 cells with OMP52M51, resistance became stable, replacing the initial unstable form of resistance seen in this model (Online Supplementary Figure S3), with possibly different underly- ing mechanisms. In the PDTALL11 model, the onset of resistance was intermediate between the other two mod- els previously described (Figure 2B). Finally, Notch path- way activation was investigated in secondary treated and non-treated resistant cells. Results disclosed marked dif- ferences in the expression of several Notch target genes in
the case of PDTALL11 and PDTALL19 and only marginal differences in the case of PDTALL8 (Figure 2C), in line with results of the first round of treatment.
Resistance to OMP52M51 is not associated with PTEN or FBW7 alterations
It is well known that the PTEN/PI3K/AKT pathway is frequently altered in T-ALL and that PTEN loss is involved in resistance induced by GSI13 and other thera- pies.14 Therefore, we analyzed the expression of PTEN in the three PDX models. PTEN was expressed in all mod- els and resistance was not associated with loss of PTEN, since the protein was detectable at comparable levels in treated and control cells (Online Supplementary Figure S4). In line with this finding, comparable levels of AKT acti- vation (determined as pAKTSer473 and pAKTThr308)15 were found in OMP52M51 compared with control cells (Online Supplementary Figure S4). Furthermore, we sequenced the FBW7 gene, since mutations in this gene have also been correlated with GSI resistance.7 Sequencing of FBW7 in PDTALL8, PDTALL11 and PDTALL19 models revealed that neither parental nor resistant cells were harboring a mutated version of FBW7 (Online Supplementary Table S2). We conclude that resist- ance to OMP52M51 did not involve mechanisms report- ed in previous studies with GSI.
Resistance to OMP52M51 is associated with a distinct transcriptional signature in PDTALL19 model
We exploited transcriptome analysis to investigate the mechanisms of resistance to OMP52M51 in the PDTALL8 and PDTALL19 models, which presented com- pletely different phenotypes of resistance. In the PDTALL8 model – the PDX characterized by late onset and stable resistance to OMP52M51 – only a few ProbeSet ID without an associated gene annotation were modulated, so we concluded that the gene expression profiles of resistant cells were superimposable to control ones (Online Supplementary Figure S5). In contrast, in the PDTALL19 model – the PDX characterized by early onset and transient resistance to OMP52M51 – we iden- tified 327 up- and 257 down-regulated genes in the com- parison between OMP52M51-resistant and control cells (Figure 3A, Online Supplementary Table S3-4). Gene set enrichment analysis (GSEA) identified 20 pathways sig- nificantly down-regulated in OMP52M51-resistant T- ALL samples (Online Supplementary Figure S6). Among them, we found the Notch pathway (Figure 3B), confirm- ing the inhibition measured by TaqMan arrays, oxidative phosphorylation, unfolded protein response, pathways related to proliferative processes - such as the G2/M checkpoint and mitotic spindle – and MYC targets, a well-known mediator of Notch activity. Interestingly, GSEA disclosed also metabolic pathways significantly down-regulated in resistant compare to control cells, including cholesterol homeostasis, adipogenesis and fatty acid metabolism (Figure 3B). Selective transcrip- tome findings were validated by quantitative RT-PCR (Online Supplementary Figure S7). In order to unravel if these modulations were due to inhibition of Notch sig- naling or were hallmarks of resistance to OMP52M51, we performed gene expression profile analysis in mice engrafted with PDTALL19 cells and acutely treated with OMP52M51. The majority of pathways identified by GSEA were present also in these samples (Online
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