Autologous T cell activation fosters ABT-199 resistance in chronic lymphocytic leukemia: rationale for a combined therapy with SYK inhibitors and anti-CD20 MoAbs

ELÍAS, ESTEBAN E.; ALMEJÚN, MARÍA BELÉN; COLADO, ANA; CORDINI, GREGORIO; VERGARA-RUBIO, MARICEF; PODAZA, ENRIQUE; RISNIK, DENISE; CABREJO MARÍA; FERNÁNDEZ-GRECCO, HORACIO; BEZARES RAIMUNDO F; CUSTIDIANO, MARÍA DEL ROSARIO; SÁNCHEZ-ÁVALOS JULIO CÉSAR; VICENTE, ANGELES; GARATE, GONZALO MARTÍN; BORGE, MERCEDES; GIORDANO, MIRTA; GAMBERALE, ROMINA

HAEMATOLOGICA

FERRATA STORTI FOUNDATION

Lugar: Pavia, Italia; Año: 2018

0390-6078

Leukemic B cells from chronic lymphocytic leukemia (CLL) patients survive and proliferate within lymphoid tissues in contact with activated T cells, myeloid cells and receiving signals through the BCR. ABT-199 (venetoclax) is a potent, selective inhibitor of BCL-2 with promising clinical activity in CLL patients. ABT-199 rapidly induces apoptosis in unstimulated peripheral blood CLL cells in vitro, while CLL cells that have received survival signals from the microenvironment are less sensitive to the drug. In particular, signaling through the BCR or signals provided by fibroblast CD40L+ and by stromal cell lines were shown to reduce the sensitivity of CLL cells to ABT-199 1-3. In this study, we investigated whether resistance to ABT-199 can be conferred by autologous T cell activation, another important survival signal from the protective niches. We first confirmed that peripheral blood leukemic cells from CLL patients are sensitive to ABT-199 in vitro in a dose dependent manner (Supplementary Figure S1), while T cells (CD3+ CD56-), NK cells (CD3- CD56+), and monocytes (CD14+) are less sensitive to the drug (Figure 1 A). In this sense, the cell death induced on CD19+ cells at 24 hours of culture with 0.01μM of ABT-199 is not even reached with a hundred times more concentrated dose of ABT-199 on accessory cells (Figure 1 A). Then, peripheral blood mononuclear cells (PBMC) from CLL patients were cultured on immobilized anti-CD3 mAbs (aCD3) to induce autologous T cell activation. As expected, aCD3 upregulated expression of the activation markers CD69 and CD25 on CD4+ and CD8+ T cells at 24 hours (not shown) and this was not affected or was only slightly reduced by the presence of clinically relevant doses of ABT-199 (Figure 1 B) suggesting that the drug does not significantly affect T cell activation. Next, we compared the impact of ABT-199 on the survival of unstimulated CLL cells or CLL cells that had received signals from autologous activated T cells. To this aim PBMC from CLL patients were incubated with or without aCD3 for 48 hours and then ABT-199 was added to cell culture. As depicted on Figure 1 C leukemic cells cultured in the presence of activated T lymphocytes were clearly less sensitive to the drug compared to leukemic cells cultured with non-activated T cells, showing that autologous T cell activation induced in vitro ABT-199 resistance in CLL cells, even at concentrations as high as 1μM. Similar results were obtained when purified leukemic cells were cultured with purified CD3+ lymphocytes from the same patient (Supplementary Figure S2 A). In line with this, we found a positive correlation between ABT-199 resistance induced in aCD3 cultures and the percentage of CD3+ cells within PBMC of each patient (Supplementary Figure S2 B). Moreover, the lesser degree of resistance to ABT-199 in Figure 1 C (see open dots in Figure 1 C) was observed with samples from Patient N° 8, 15 and 17 which had very low percentages of T cells within PBMC (see Supplementary Table S1), while no other clinical or biological association was found. Since CLL patients generally have higher absolute numbers of CD3+ cells4 which are mainly localized within proliferation centers of lymphoid tissues, where activated T cells tend to cluster around proliferating CLL cells5, 6, our results suggest that tissue resident leukemic cells might not be properly targeted by ABT-199. The activation of autologous T cells increased the expression of the activation marker CD86 in CLL cells in all of the patients evaluated (Figure 1 D) and, more interestingly, it induced the upregulation of the antiapoptotic proteins MCL-1 and BCL-XL on CLL cells (Figure 1 E). The fact that these proteins from BCL-2 family are not targeted by ABT-199 may explain the resistance induced by activated T cells, which was also suggested by recent studies analyzing other microenvironment signals 1-3. In an attempt to restore ABT-199 sensitivity in CLL cells, we employed the BCR kinase inhibitor (BCR-KI) GS-9973 (entospletinib) which inhibits SYK and is an attractive novel therapeutic agent for CLL7. As it is shown in Figure 1 F, we found that a clinically relevant concentration of GS-9973 (1μM) was able to reduce the resistance to ABT-199 induced by activated T cells. We recently reported that GS-9973 thwarts T cell activation in CLL patients not only impairing the expression of CD25 and CD69 on CD3-stimulated T cells, but also reducing the proliferation and the cytokine production by T cells 9. Moreover, when the direct effect of GS-9973 on the survival of CLL cells was evaluated, we found that it only slightly reduces leukemic cell survival but not in a statistically significant way, and does not particularly sensitize CLL cells to ABT-199 induced cell death (Supplementary Figure S3). Altogether, our observations suggest that GS-9973 was able to reduce the resistance to ABT-199 induced by activated T cells probably by impairing T cell activation.Our findings strengthen the role of the tumor microenvironment in the promotion of drug resistance, which might account for the progression of the disease in ABT-199 monotherapy-treated CLL patients10. They also encourage the combination of ABT-199 with BCR-KIs that impair activation signals provided by the tumor microenvironment8. In the case of GS-9973, while we found that it overcomes the resistance to ABT-199 induced by activated T cells (Figure 1 F) others reports showed that it prevents BCR mediated resistance of CLL cells to the drug more efficiently than other BCR-KIs 2.Given that the triple combination of ABT-199 with BCR-KIs and anti-CD20 mAbs appears as an attractive strategy (see Clinical trial number NCT03379051, NCT02950051, NCT02758665, NCT02296918 and NCT02427451 at www.clinicaltrials.gov) we aimed to determine whether ABT-199 may affect phagocytosis of rituximab-coated CLL cells, which is a central mechanism in the anti-tumor activity of anti-CD20 antibodies11, 12. Macrophages were exposed to different doses of ABT-199 and then, CFSE-labeled CLL cells or rituximab-coated CFSE-labeled CLL were added to the culture. Phagocytosis was evaluated after 1 hour. Preincubation of macrophages with ABT-199 for 3 hours (Supplementary Figure S4) or 18 hours (not shown) did not modify their capacity to uptake uncoated or rituximab-coated CLL cells. On the other hand, preincubation of purified CLL cells with ABT-199 significantly increased their uptake by macrophages, not only when coated with rituximab but also in its absence (Figure 2 A and B). This interesting finding could be due to the exposure of ?eat-me signals? on dying CLL cells which prompts macrophages to engulf them13. Given that phosphatidylserine (PtdSer) exposure during apoptosis is one of the best characterized ?eat-me signals? 13, we evaluated Annexin V binding to PtdSer on purified CLL cells incubated or not with ABT-199 for 3 hours. Results from Figure 2 C confirm increased Annexin V binding in ABT-199-treated CLL cells. Representative dot plots are shown in Figure 2 D. By contrast, rituximab binding to CLL cells was not affected or even seems to be reduced by the drug (Figure 2 E). Representative histograms of rituximab binding are shown in Figure 2 F. The enhanced phagocytosis of rituximab-coated ABT-199-treated CLL cells might account for the substantial benefit showed in CLL patients treated with ABT-199 and rituximab compared with ABT-199 monotherapy 14.Finally, to test whether the stimulatory effect of ABT-199 on phagocytosis also functions in the presence of GS-9973, CLL cells were pre-incubated with or without ABT-199 as mentioned above and phagocytosis was performed with macrophages treated or not with GS-99739. We found that GS-9973 does not modify the uptake of uncoated CLL cells by macrophages (Figure 2 G). Moreover, we confirmed our previous data showing that GS-9973 reduced phagocytosis of rituximab-coated CLL cells9 and found that it also impairs the phagocytosis of ABT-199 treated-CLL cells coated with rituximab. However, ABT-199 fostered uptake of rituximab-coated CLL cells in the presence of GS-9973 (Figure 2 G). In conclusion, the resistance observed in vitro when CLL cells were cultured with autologous activated T cells suggests that leukemic cells from the supportive microenvironment might not be properly targeted by ABT-199 monotherapy. Our results encourage the combination of the drug with BCR-KI, such as GS-9973 which overcame the resistance induced by activated T cells and by other microenvironment signals2. Combination with CD20 mAbs could also be useful because ABT-199 enhanced phagocytosis of rituximab-coated-CLL cells, even in the presence of GS-9973.