Page 167 - Haematologica May 2020

Resistance to Notch1 neutralizing antibody
found that tumor adaptation to anti-NOTCH1 therapy was accompanied by a distinct transcriptomic and meta- bolic signature. Alterations converged on lipid metabo- lism, hinting at significant alterations in constituents of cell membranes such as phosphatidylcholines, lysophos- phatidylcholines, sphingomyelins and ceramides. Moreover, there was a trend towards the reduction of the unsaturation degree of lipids in OMP52M51-resistant samples, hypothetically suggesting a modulation of membrane fluidity, since saturated fatty acids are more densily packed.21 Some years ago, a well-designed study correlated a different composition of phospholipids with an increase in endocytosis and therefore a reduction in EGFR and Notch signaling, due to the fact that these receptors were less abundant on the cell membrane.22 Albeit performed in Drosophila, we speculated that the different composition and/or unsaturation degree of membrane components could affect the amount of NOTCH1 receptor on cell surface and, therefore, the amount of target available for binding the therapeutic antibody. For a linear pathway such as Notch, the expo- sure of the receptor on cell surface is fundamental and, therefore, the number of NOTCH1 receptors are strictly regulated.23-25 However, we found reduced NOTCH1 lev- els also after short-term treatment with OMP52M51, a condition where lipid alterations were not detected, indi- cating that the alterations in lipid metabolism are in fact a feature associated with the resistance in the PDTALL19 model, but this does not likely cause reduced NOTCH1 FL levels in these cells. In any case, recent findings gave prominence to new functions of membrane components, beyond the structural one. Phosphatidylcholines, lysophosphatidylcholines, sphingomyelins and ceramides could, in fact, be involved in cell signaling and behave as messengers frequently altered in pathologic conditions such as cancer.26-29 Therefore, it could be pos- sible that the variations in membrane components observed in PDTALL19 model could influence the microenvironment and/or T-ALL cell behaviour. Along this line, our finding that T-ALL cells resistant to OMP52M51 had reduced size and showed lower surface levels of CD3, CD4 and CD11a compared with control cells supports this hypothesis.
In a recent paper, Herranz and colleagues identified glucose metabolism and glutaminolysis as key determi- nants in cell resistance to Notch blockade with GSI.30 In our model, we also found an increase in the amount of hexoses in resistant cells, but the innovative finding was represented by the association between the resistance to OMP52M51 antibody and the modulations of the sterol biosynthetic pathway. Moreover, in our model we did not observe the activation of autophagy (data not shown), which other groups previously detected in cells after Notch inhibition by GSI.30 Regarding the PDTALL19 model, finally, the comparison between resistant cells and acute treatment with OMP52M51 disclosed that the anti- body inhibited the same GSEA pathways but with a dif- ferent strength.
Another possible hypothesis behind the reduction of surface NOTCH1 receptor regards DELTEX1. Many stud- ies performed in Drosophila have shown that the activa- tion, degradation and recycling of Notch receptors could
depend on their ubiquitination pattern.31,32 DELTEX1 is an ubiquitin-ligase that could act as a positive or a negative regulator of Notch signaling depending on the cellular context and could influence endocytic trafficking of the receptor.33-35 Notably, in our models DELTEX1 was one of the most down-regulated genes in PDTALL19 cells, whereas it was not detectable in PDTALL11 cells and was not modulated in PDTALL8 cells (Figure 1C), PDX in which we did not observe any reduction of surface NOTCH1 protein.
In the PDTALL8 model, given the stability of the resist- ance and the loss of Notch signaling inhibition, the most likely explanation for the observed resistance was the selection of a mutated clone no longer responsive to ther- apy. To address this possibility, NGS analysis performed in control and resistant cells disclosed a selection of clones of the initial tumour, since treated samples pre- sented a lower mutational burden compared to control. Moreover, NGS analysis revealed the presence of two activating mutations in NOTCH1 gene - p.Q1584H and p.L1585P - in the OMP52M51 resistant mice. These vari- ants, reported as T-ALL associated in CIViC database (36), were in cis and cause the introduction of a positive charge (p.Q1584H) and a break (p.L1585P) to the α2-helix in the HD domain,16 adding steric bulk in the HD core. These modifications potentially affect the stability of HD domain and could influence binding of the OMP52M51 antibody, causing the loss of responsiveness to the thera- py. Notably, we confirmed the p.Q1584H and the p.L1585P mutations by targeted sequencing but could not find them in the parental cells prior to treatment. However, as all of the independently derived resistant PDTALL8 tumor samples disclosed the same p.Q1584H and p.L1585P variants, it is extremely likely that these variants must have been present in a minor sub-clone prior to selective pressure with OMP52M51.
Finally, the possible role of the microenvironment must be taken into consideration, since different tumours could differ, not only in intrinsic lesions but also in their dependence on the niche – therefore, different T-ALL cases might respond distinctly to the same treatment. A better characterization of both cell-autonomous lesions and cues from the microenvironment, which was apart from the purpose of this paper, would extend the com- plete understanding of how the malignancy progresses and resistance develops.1
In conclusion, our study highlights heterogeneity in the phenotypic and molecular features of resistance to OMP52M51. Notably, traits associated with resistance differ from those previously described following treat- ment with GSI, suggesting that leukemia cells can adopt several strategies to evade Notch inhibition according to the therapeutic drug used.
Ackowledgments
We thank Dr. Erich Piovan (University of Padova, Padova, Italy) for providing the PTEN antibody and Dr. Tim Hoey (Oncomed Pharmaceuticals Inc., Redwood, CA, USA) for pro- viding the OMP52M51 antibody.
Funding
This work was supported by AIRC (Grant n. IG18803 to SI).
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