Page 304 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
castle Hematology Biobank, after appropriate consent (reference numbers 2002/111 and 07/H0906) from children treated with a with a standard three or four drug induc- tion ALL regimen and who had slow clearance of disease (MRD levels ranging from 0.30% to 56.70% with a mean of 19.99% +/- standard deviation [SD] of 18.50). We analyzed 14 bone marrow samples at presentation of T-ALL and eight samples from five patients during follow-up, taken at day 8 (N=3), day 21 (N=1) and day 28 of induction therapy (N=4) using mass cytometry with previously published methods (antibody panel is shown in Online Supplementary Table S1).1 Patient details are shown in Table 1.
Live, single, non-apoptotic ALL cells were identified by their specific LAIP and the mean mass intensity (MMI) of all antibody signals expressed relative to those from normal CD4-positive cells present in the same sample. The most prominent signals in presentation samples were pCREB/ pATF1 [S133] (mean 214.55; range, -50.82 to 466.60), Ki67 (mean 158.97; range, 0.67-582.60) and pSRC family [T416] (mean 80.44; range, -43.90 to 344.16) (Figure 1A; Online Supplementary Table S2). The latter having binary separation with high and low activation and likely to reflect levels of pLCK.2,3 Western blotting confirmed that the prominent activated form was pCREB, with minimal signal from pATF1 (Online Supplementary Figure S1). Compared to presen- tation ALL cells, MRD cells had a significant decrease in Ki67 levels with a mean of 158.97 falling to 34.62 and an increase in pHH3, with a mean of 21.20 increasing to 87.91 in MRD (P<0.05) (Figure 1B; Online Supplementary Table S2). There were also more subtle changes in pSTAT5, a signal of 2.96 at presentation decreasing to 1.27 at MRD and an increase in pNFKB p65, -0.30 at presentation increasing to 2.06 in MRD cells (P<0.05).
We next used cytometry by time of flight (CyTOF) workflow R pipeline to perform cluster analyses using phospho-signals (and Ki67) from ALL cells to investigate cluster composition in drug naïve cells and in those after therapy. We generated 20 unique ALL cell clusters (Figure 1C, D; Online Supplementary Table S2). ALL populations were complex heterogeneous, showing multiple different clusters at both presentation and in MRD cells, but there were clear differences in cluster abundance (Figure 1C). The closely related clusters, 4 and 5, as well as cluster 18, were significantly more represented at presentation (P<0.05) and were characterized by multi- ple signaling nodes including pLCK, pCREB, pRb, pp38 and high Ki67. There were a number of clusters more prevalent in MRD cells including clusters 1, 12, 13, 16 and 17 (P<0.05). All of these MRD clusters had strikingly low levels of Ki67, with the more abundant ones also expressing higher lev- els of pHH3 and pRb. Uniform manifold approximation and projection visualization of MRD and presentation clusters revealed MRD to be enriched in the left and right hand ar- eas of the map. Visualization of pCREB confirmed its almost ubiquitous expression across the UMAP in both presentation
and MRD ALL cells. Clusters 1 and 6 were exceptions in that they expressed low pCREB but represented less than 1% of all cells but cluster 1 was more prominent in MRD (Fig- ures 2A, B and 1C, D). Visualization of Ki67 clearly showed MRD-enriched areas to have significantly lower expression (Figure 2C; Online Supplementary Figure S3). This was even more apparent in UMAP projections from individual patients (Figure 2D) and, for patients with multiple time points, there was a further selection of Ki67-negative cells as treatment progressed (LK287, LL223 and LK350). Notably, only one of five patients had MRD cells had both Ki67-negative and positive cell islands (LK360). Clusters 12, 13, 16 and 17 were visible in presentation samples but were clearly more prominent in the MRD (Figure 2E, F).
We next assessed the sensitivity of four patient-derived xenografts (PDX) T-ALL co-cultured with mesenchymal stromal cell support to 666-15, a selective CREB inhibitor, and showed a mean ± sd, Half maximal inhibitory concen- tration (IC50) of 3.6±0.2 μM (Figure 2G). Robust, dose-de- pendent apoptosis was shown with 666-15 (N=3; P=0.01) with almost 100% of cells in the early stages of apoptosis after 48 hours exposure to IC50 concentrations (Figure 2H). At the same time point, cell cycle analyses corroborated the cytotoxic action of 666-15 by the increased proportion of cells with subG1 DNA content, though this did not attain statistical significance (N=3; P=0.07), and also demonstrat- ed the majority of cells to be in G0/G1 (Figure 2I).
The advancement in cytometric methodologies has pro- vided a stepwise increase in the number of analytic pa- rameters that are enabling extensive functional analyses of rare cell populations, such as clinical MRD. Here, we confirm the presence of activated CREB in almost all T ALL cell clusters at both presentation and MRD and show that MRD is non cycling and, similar to B ALL, is derived from cell populations evident at diagnosis that are en- riched during induction therapy. Dormancy is a common mechanism of cancer cells to achieve/acquire multi-drug resistance, at least to therapies reliant upon cell division,4 and our data support others in suggesting that ALL MRD cells are dormant.5 Eliminating MRD cells at the end of induction might be best achieved with targeted drugs that are not dependent on cycling cells such as the CREB pathway or alternatively re-engaging cell cycling, prior to more standard chemotherapeutics.
CREB overexpression and hyperactivation is reported in a number of cancer types and is considered a therapeutic target as it appears to be a vital signallng node down- stream of many oncogenes.6 An earlier study by van der Sligte et al., demonstrated that both knockdown and pharmacological inhibition of CREB induced cell cycle arrest and apoptosis in T ALL cells by impacting on genes involved in apoptosis, cell cycle and glucose metabo- lism.7 Here, we confirm the dependency of T-ALL cells on CREB activity with a next generation CREB inhibitor,
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