B-Precursor Acute Lymphoblastic Leukaemia Causes Paracrine Senescence in Mesenchymal Stromal Cells to Subvert the Treatment Response in Adult Acute Lymphoblastic Leukaemia

We previously showed that both reactive oxygen species-inducing chemotherapy and some genetic subtypes of B precursor acute lymphoblastic leukaemia (ALL) can induce bone marrow (BM) mesenchymal stromal cells (MSC) to become cancer associated fibroblasts (CAFs). Exposure of MSC to ALL-cell conditioned media (CM) is sufficient to generate CAFs - direct cell-cell contact is not required (Burt, Dey et al, 2024). We showed that CAFs can lead to chemoresistance via transfer of mitochondria to ALL cells through tunnelling nanotubes (Burt, Dey et al 2019). We noted that CAFs in the ALL BM had features of senescence, prompting the current study in which we aimed to investigate the link between senescence and CAFs in ALL. We used 3 genetic subtypes of ALL; BCR::ABL1 (SD1 cells, representing “adult” ALL) , ETV6::RUNX1 (REH cells, representing “childhood” ALL) and KMT2A::AFF1 (SEM cells, known from our previous work NOT to induce CAFs). We exposed HS27a stromal cells to the ALL CM at a number of timepoints over a period of three weeks and characterised senescence using qPCR, cytochemical B-galactosidase assays, flow cytometry and immunofluorescence (IF). Cytokine arrays were performed to quantify the senescence associated secretory phenotype (SASP) components and assess the complexity of SASP components in all three ALL subtypes. A more prolonged exposure (> 10 days) to CM was required for senescence to develop in contrast to CAF formation which occurs early (2-3 days) in HS27a MSC cells. Senescence marker expression peaked at around day 11 to day 14 of CM exposure before subsiding to lower levels around day 21. On day 14, 70% of HS27a cells treated with SD1 CM were positive for beta galactosidase compared to below 25% positive for REH and SEM cells. Cell cycle arrest was demonstrated by P16 and Ki67 IF. 95% of SD1 CM treated HS27a cells were positive for P16 compared to less than 10% in RPMI control. Likewise, only 29% of HS27a exposed to SD1 CM were Ki67 positive compared to 66% in the RPMI control. Expression levels of other senescence-associated markers such as SERPINE1, SERPINB2, SOD2, NFKB1 were quantified by qPCR and were all significantly (p<0.001) upregulated in SD1 CM treated HS27a (2 to 40 fold) compared to SEM CM (-2 to 5 fold) and REH CM (-0.5 to 1.0) treated HS27a. SASP associated cytokines IL6 (1000-5000pg/ml), IL8 (2500-5000pg/ml), CXCL10 (500-1500pg/ml), CCL5 (1500-2250pg/ml), IL10 (25-50pg/ml), were significantly (p<0.0001) upregulated in the SD1 CM treated HS27a compared to controls and other ALL cell line exposed HS27a. Finally, HMGB2 and LMNB1 genes, loss of which is associated with senescence were significantly (p<0.001) downregulated in the SD1 CM (-2.5 to 0.5 fold) treated HS27a compared to controls.

In view of the prominence of senescence in SD1-exposed HS27a cells, we tested the effects of two different senolytic drugs venetoclax (BCL2i) and navitoclax (BCL2i/BCLXLi) in vitro. Briefly, HS27a cells were exposed to SD1 CM for 11 days, and after washing, increasing concentrations of the drugs were added for up to 72 hrs followed by washing, fixing and DAPI-staining and counting. HS27a exposed to SD1 CM was highly sensitive to low dose treatment of navitoclax (0.5uM) with 80% cell death 48 hrs post treatment. HS27a cells exposed to SD1 CM were less sensitive to venetoclax with only 50% cell death observed at higher doses (5uM) even after 72 hrs post treatment. Further senolytic drug screening experiments are ongoing.

Our data suggest that BCR::ABL1 but not ETV6::RUNX1 or KMT2A::AFF1 ALL cells, can induce paracrine senescence in HS27a MSC cells. We suggest that paracrine senescence in the stromal microenvironment may be a component of treatment response in B-ALL.

^^ and ** contributed equally

Innes:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Conference fees, Speakers Bureau; Incyte: Speakers Bureau. Fielding:Autlous: Other: payment for advisory board attendance; Incyte: Other: payment for advisory board attendance; Amgen: Other: payment for advisory board attendance, funding contribution towards hosting an academic meeting.