The spleen as a site for residual disease in T-cell ALL

T-cell acute lymphoblastic leukemia (ALL) is highly aggressive and frequently spreads from the thymus to the bone marrow, liver, lymph nodes, and spleen among other areas. A subtype of T-cells ALL, known as early T-cell precursors (ETPs) have been associated with a poor prognosis and high risk of relapse. ETP-ALL has been shown to be dependent on B-cell lymphoma-2 (Bcl-2) for survival, and as a result is sensitive to treatment with the BH3 mimetic ABT-199.

While targeted therapies such as ABT-199 can be very effective, acquired resistance is a major problem. A review published in Blood Advances by Alessandra Di Grande and colleagues, investigated the spleen as a site of residual disease after ABT-199 treatment.¹

Residual disease in the spleen in a LOUCY xenograft model following ABT-199 treatment

Using a xenograft murine model with an ETP-ALL-like, green-fluorescent protein/luciferase-labeled, LOUCY cell line, which has been shown to be sensitive to ABT-199, the in vivo response to treatment with this agent was investigated. To monitor response, bioluminescent imaging was used to measure leukemic burden.

After treatment with ABT-199, there was a significant decrease in whole-body flux measurements (p ≤ 0.0001) along with a reduction in hCD45⁺ cells in the peripheral blood. However, while the whole-body flux measurements decreased overall, an increase in flux of the spleen was seen in the ABT-199-treated mice compared with placebo-treated mice. These results suggest the spleen is the preferred sanctuary site for residual leukemia cells.

Evidence for splenic microenvironment changing Bcl-2 dependence in murine studies

Leukemic cells from the spleen of the LOUCY xenograft model mice treated with ABT-199 or vehicle were isolated following 2 weeks of treatment, along with LOUCY cells cultured in vitro. BH3 profiling showed the splenic ABT-199-treated cells had a reduced response to the BAD BH3 peptide along with ABT-199 compared with the in vitro LOUCY cells. These data indicate a decrease in Bcl-2 dependence in the splenic microenvironment.

The gene and protein expression of Bcl-2 was examined next, and it was found that expression was decreased at both the mRNA and protein level in cells isolated from the murine spleens compared with the in vitro cell line (p ≤ 0.0001).

Residual disease in the spleen in ETP-ALL patient-derived xenograft model following ABT-199 treatment

A patient sample-derived xenograft model was made using ABT-199 sensitive tissue. Mice were treated with vehicle or ABT-199 (25 mg/kg) daily for up to 34 days. Endpoint analysis showed significantly reduced hCD45⁺ cells in the bone marrow (p ≤ 0.0001) and blood (p = 0.0014) of the ABT-199-treated animals compared with the vehicle-treated mice. Residual disease (hCD45⁺ = 15%) was seen in the spleens of the ABT-199-treated animals in this model (p < 0.0001).

Mitochondrial priming of bone marrow and spleen cells from the patient-derived xenograft model using BH3 profiling was performed. The leukemic blast cells recovered from the spleen showed a decreased response to the BIM peptide compared with cells from the bone marrow, indicating a reduced sensitivity to apoptotic signaling caused by the splenic microenvironment.

Human splenic microenvironment influences Bcl-2 dependence

A human splenic fibroblast (HSF) line was used to represent the spleen and cultured with LOUCY cells for 48 hours. Cytochrome c release was significantly reduced following treatment with BAD BH3 peptide and ABT-199. Reduced sensitivity to ABT-199 (treated for 24 hours) was also recorded after a 48-hour incubation of the LOUCY cells with the HSF. Bcl-2 expression was downregulated at the protein and mRNA levels following a 24-hour co-culture with HSFs.

Transwell migration assays with conditioned media from HSFs, and human bone marrow fibroblasts were used to assess if LOUCY cells migrated to the spleen preferentially. LOUCY cells were found to be one-fold higher in the conditioned media-HSFs compared with the bone marrow fibroblasts (p = 0.0006).

To evaluate whether co-culture of human splenic fibroblasts with and ETP-ALL sample would change the BH3 profile, cells were cultured together for 16 hours. In the ETP sample BAD BH3 peptide response was decreased by 20% following co-culture, showing reduced Bcl-2 dependence. These results indicate that the spleen may generate positive migratory signals and that co-culture with HSFs can reduce Bcl-2 dependence in the LOUCY cell line and patient-derived sample.

Sequencing of LOUCY cells following treatment with ABT-199

Single-cell RNA sequencing of an additional LOUCY xenograft model was performed to assess the impact of ABT-199 treatment in the spleen and bone marrow. For this model, the mice were treated with ABT-199 (50 mg/kg) or vehicle for 11 days. A potential relapse of disease was recorded in this model, with splenomegaly and an increase in hCD45+ cells in the blood being recorded for both the vehicle and ABT-199-treated mice.

After analysis, 87 genes were upregulated and 132 were downregulated in LOUCY cells from the spleen and bone marrow of mice treated with ABT-199.

Of these:

• 117 were differentially expressed in the spleen
• 166 were differentially expressed in the bone marrow

Following statistical analysis, 18 genes were found to be significantly upregulated while 35 were significantly downregulated in the spleen.

T-cell differentiation markers in the spleen of treated mice

Gene ontology was used to assess which pathways were altered between the spleen and the bone marrow. In the spleen, T-cell differentiation was the most upregulated process, whereas in the bone marrow, negative regulation of cell proliferation and positive regulation of transcription from RNA polymerase II promoter, were the most upregulated processes.

Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis showed a significant increase in genes associated with:

• cellular senescence in the spleen
• fluid shear stress and p53 signaling in the bone marrow

Transcription factors associated with T-ALL demonstrated changed expression following ABT-199 treatment.

• LCK and TCF7 were downregulated
• c-MYB and GATA3 were upregulated

Both GATA3 and c-MYB are important for T-cell differentiation. These results suggest that the spleen microenvironment is a possible site of T-cell differentiation, unlike the bone marrow.

Generation of ABT-199 resistant model to validate findings

ABT-199 resistant LOUCY cells were generated, and these cells were shown to have reduced Bcl-2 expression at the mRNA and protein level.

From the RNA sequencing results, Di Grande and colleagues hypothesized that ABT-199 resistance may be a consequence of the selection of a more differentiated blast cell. The expression of differentiation markers CD1a (p = 0.0074) and surface CD3 (p = 0.029) were found to be significantly increased in the ABT-199 resistant cells compared with the parent LOUCY cell line. Therefore, resistance to ABT-199 may be through selection of a more differentiated blast.

Conclusion

This study demonstrated that in the LOUCY xenograft murine model of T-ALL, the spleen acted as a site for residual disease. The splenic microenvironment alters the dependence on Bcl-2, both in LOUCY cells and the patient ETP-ALL cells. In addition, the results of this study suggest that the mechanism of resistance to ABT-199 treatment involves differentiation to a more mature form of T-ALL.