Targeting the dysfunctional mitochondrial respiration in drug resistant B-cell acute lymphoblastic leukemia

Reprogramming of cellular pathways is a crucial mechanism of drug resistance and survival in refractory acute lymphoblastic leukemia (ALL) cells. In the present study, we performed an unbiased gene expression analysis and identified a dysfunctional mitochondrial respiration program in drug-resistant ALL cells grown in a co-culture system with bone marrow stromal cells (BMSC). Specifically, the activity of the complexes within the electron transport chain was significantly downregulated, correlated with decreased mitochondrial mass and ATP production in drug-resistant ALL cells. To validate mitochondrial respiration as a druggable target, we utilized pyrvinium pamoate (PP), a known inhibitor of mitochondrial respiration and documented its anti-leukemic activity in several ALL cell lines grown alone or in co-culture with BMSC. To increase the bioavailability profile of PP, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. PP anti-leukemic activity was decreased by the addition of sodium pyruvate, and furthermore, PP was found to have an additive anti-leukemic effect when used in combination with rotenone, a mitochondrial complex I inhibitor with activity similar to PP on the mitochondrial respiration. Importantly, PP’s cell death activity was found to be specific for leukemic cells as primary normal immune cells were resistant to PP-mediated cell death. In conclusion, we have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells.

141 Electron transport chain complex activities 142 The activity of the ETC Complexes I, III, IV and V were measured as previously 158 Mitochondrial staining assay 159 Leukemic cell populations were isolated from long-term co-culture as described above.  248 Specifically, Complex I activity was decreased by 58 % in S cells and by 55 % in the PD 249 cells as compared to M cells. Interestingly, for SUPB15 cells, the PD cells showed 250 significant reduction in Complex III activity compared to M and S cells (Fig. 2B).
12 251 Furthermore, Complex IV and Complex V showed no significant differences in activity 252 between the three cell populations in either REH or SUPB15 cells ( Fig. 2C and D).

Leukemic cells in co-culture have dysfunctional mitochondrial bioenergetics 254
To understand how the decreased ETC complex activity of the leukemic cells in co-255 culture affects the mitochondrial bioenergetics, we looked at ATP production and the 256 relative mitochondrial mass within the different sub-populations of leukemic cells in co-257 culture. We found that the ATP production was significantly decreased in both REH and 258 SUPB15 cells in co-culture with BMSC when compared to the M cells ( Fig. 3A and B). 284 in the PD cell population (Fig. 4F).

286
Since PP has poor bioavailability, we encapsulated it within nanoparticles composed 287 of PGLA and PEG complexes. We first determined if the nanoparticles (NP) by 288 themselves had any activity on the bone marrow stromal components or the ALL cells.
289 As shown in Fig. 5A, NP at doses as high as 100 M failed to elicit any death in BMSC, 290 osteoblasts (HOB), NALM27 or REH ALL cells lines. Next, we tested the toxicity of the 291 PP encapsulated in NP in normal cells compared to ALL cells. Fig. 5B shows that 250 nM 292 of the PP encapsulated in NP did not have any deleterious effects on BMSC, normal bone 293 marrow mononuclear cells (BMMC), or normal peripheral blood mononuclear cells 294 (PBMC). However, it did cause a significant decrease in cell proliferation (46 %) when 295 exposed to a B lymphoblastoid cell, SD1 (Fig. 5B). To ascertain that the differential effects 296 of PP between the normal and the leukemic cells was not due to differences in cell 14 297 proliferation rate, we utilized normal primary CD3+ cells and induced proliferation using a

CD3/CD28 T cell activator. Treatment of dormant or proliferating T cells with PP did not
299 result in decreased cell viability in the dormant T cell population and showed a minimal 300 decrease in viability (~13 %) in the proliferating T cells (Fig. S4). Finally, we wanted to 301 determine the anti-leukemic effects of the PP encapsulated NP in a hemosphere assay.
302 We utilized SD1 cells to form hemospheres for 4 days, after which they were treated with 303 250 nM of the PP encapsulated NP. After 48 hrs of treatment, the NP were able to 304 penetrate the pre-formed spheres resulting in a loss of hemospheres (Fig. 5C).
305 Quantitation of the spheres using light microscopy showed that the PP encapsulated 306 nanoparticles resulted in a loss of spheres and were efficient in inhibiting the sphere-307 forming abilities of SD1 cells, to the point no spheres formed after treatment (Fig. 5C).

309
To understand the mechanism of the PP-induced cell death in ALL cells, we analyzed 310 its effect on ATP production. As seen in Fig. 6A  326 PP and rotenone induced an additive 40% cell death (Fig. 6F).

328
In the present study, we have utilized an in vitro co-culture system to characterize PD

381
We had previously shown, using a Seahorse metabolic assay, that rotenone induced 382 a complete inhibition of oxygen consumption rate in ALL cells [10]. In our study, 383 pretreatment of ALL cells with rotenone did not abolish the anti-leukemic activity of PP.