Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques — BH3 profiling and high-throughput kinase activity mapping — we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.


B) C) D)
A) For a more detailed description of the peptide sensors design, sequence and connectivity between peptides and kinases, as well as data normalization steps and analysis, refer to: (3,5).
Here, we used HT-KAM as a modular peptide-sensor platform to specifically interrogate the activity of kinases that can affect the phosphorylation and stability of BCL-2 family proteins, i.e., we chose to measure and compare the activity of kinases belonging to the MAPK, PKA, PKC, PKD, CDK and AMPK families (the complete list of kinases and kinase families is provided in Fig. 4E).
To prepare protein extracts to run on the HT-KAM platform, cells were washed three times with cold PBS and lysed with freshly prepared 1X cell lysis buffer (#9803, Cell Signaling Technology) complemented with 1X of Halt Protease & Phosphatase (#1861281, Thermo Fisher Scientific).Cell lysates were collected and spun down at 14,000rpm for 15min at 4ºC and supernatants stored at -80ºC.

ii) Trypan Blue exclusion assay
Live and dead drug-treated cells were measured using the Trypan blue stain (#T10282, Invitrogen) and Countess® II cell counter (#AMQAX1000R, Invitrogen).
iii) Annexin V/Hoechst assay Stroma NKTert cell co-culture was used to sustain survival for primary cells at a 1:10 (NKTert:CLL) ratio.NKTert cells were first seeded in 24-well plate 24 hours prior to addition of primary cells and drug treatment.Following drug treatment, cells were harvested and seeded in 96-well flat-bottom plates and stained with Annexin V and Hoechst staining in 1x Annexin-V binding buffer (100 mM HEPES, 40 mM KCl, 1.4 M NaCl, 7.5 mM MgCl2, 25 mM CaCl2 pH 7.4) at room temperature (RT) for 15 minutes.Samples were then fixed with Annexin V fix buffer (4% parafolmaldehyde, 0.5% glutaraldehyde in 1X Annexin V binding buffer) at RT for 10 minutes and neutralized with N2 buffer (1.7 M Tris, 1.25 M glycine, pH 9.1) prior to analysis using the BD FACS Fortessa flow cytometry as previously described (6).Stroma NKTert cells were not used for cell lines.Fisher Scientific) as previously described (6,7).

Co-immunoprecipitation (Co-IP)
Cells were lysed in Pierce TM IP buffer (#87788, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (#A32961, Thermo Fisher Scientific).Cell lysate containing 1-1.5mg proteins were transferred for pre-clearing using protein agarose A (#sc-2001, Santa Cruz Biotechnology) or G (#sc-2002, Santa Cruz Biotechnology) beads on a rotator at 4°C for 1.5 hours.Supernatant from this pre-clear was then transferred to a new 1.5 mL Eppendorf tube and 3µg IP antibody was added to sample for overnight incubation on a rotator at 4°C. 25µL protein agarose A or G beads were then added to sample for pull-down on a rotator at 4°C for 6 hours.Sample was then washed with Pierce TM IP buffer 3 times before 25µL of loading dye (#7722, Cell Signaling Technology) was added to the sample and heated at 95°C for 15 minutes for Western blot analysis.

Supplemental Figure 10 .
viability of 9 healthy donor normal primary cells (HC, n=9), consisting of PBMC (n=3), B cells (n=3) and T cells (n=3) and primary CLL cells (CLL, n=16) following pre-treatment with FTY720 (1µM) for 4 hours followed by co-treatment with venetoclax (10nM) for 24 hours, measured by Annexin/Hoechst assay.Sidak's multiple comparisons tests was used.Some DMSO and venetoclax mono-treatment control samples of primary CLL used in this experiment were shared with those used for the cell viability experiment done in figure12Ddue to limited patient materials.B-D) Cell viability of primary cells (same 9 samples from figureS9A), divided to their respective types, PBMC (n=3), B cells (n=3) and T cells (n=3) following pre-treatment with FTY720 (1µM) for 4 hours followed by co-treatment with venetoclax (10nM) for 24 hours, measured by Annexin/Hoechst assay.Sidak's multiple comparisons tests was used.Ex vivo treatment with FTY720 reduces BCL-2 family protein phosphorylation via PP2A activation and subsequently affect the pro-and anti-apoptotic protein interaction in treatment-naïve CLL patient samples.A) Quantified normalized expression values derived from T163pMCL-1/b-Actin, S70pBCL-2/BCL-2 and MCL-1/b-Actin following ex vivo pre-treatment with OA (5nM) for 2 hours followed by co-treatment with FTY720 (2.5µM) for 4 hours in CLL cells for figure 12F.Bands quantified by ImageJ software.Sidak's multiple comparisons tests were used.B) Densitometry analyses of % normalized BAX/BCL-2 pull-down following ex vivo 4-hour pre-treatment with FTY720 (2.5µM) and 1-hour co-treatment with ABT-199/VEN (10nM) in CLL cells for figure 12G.Dunnett's multiple comparisons test was used.cytochrome c release, which indicates specific dependences for anti-apoptotic protein, by HRK is measured for BCL-xL, MS1 for MCL-1, BAD for BCL-2 and/or BCL-xL and venetoclax for BCL-2.BIM was used as overall mitochondrial priming for apoptosis.Kinase activity mapping assayTo measure the activity of kinases in biological samples, we used a biochemical assay named high throughput kinase-activity mapping (HT-KAM) platform(3).HT-KAM uses libraries of peptides as sensors of the phosphorylation activity of kinases(3).The activity of kinase enzymes is derived from their respective subset of biological peptide targets included in the assay.Peptides are derived from computationally curated biological targets of kinases' substrates deposited in PhosphoAtlas (4).The phospho-catalytic signature of cell extracts is established from simultaneously occurring ATP-consumption tests measured in the presence of individual peptides that are experimentally isolated from each other.Assays are run in 384 or 1536 well-plates handled by automated liquid-dispensing instruments.Each experimental well contains one peptide (200µg/ml 11-mer peptide), and all wells receive kinase assay buffer (#9802, Cell Signaling Technology), ATP (#9804, Cell Signaling Technology), cell sample (prepared from cell at ~10µg/ml total protein extract).All reagents are kept on ice and plates on cold blocks until enzymatic reactions are started.Once the dispensing of the reaction mixtures is complete, plates are incubated for 1h at 30ºC.ATP is detected using Kinase-Glo revealing reagent (#V3772, Promega), which stops the activity of the kinases and produces a luminescent signal that directly correlates with the amount of remaining ATP in the samples.

Figure diagrams were generated
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