Venetoclax and dexamethasone synergize with inotuzumab ozogamicin–induced DNA damage signaling in B-lineage ALL

Adult patients with relapsed acute lymphoblastic leukemia (ALL) have a dismal prognosis with cure achievable only by allogeneic stem cell transplantation or chimeric antigen receptor T-cell therapy.^1-4  Inotuzumab ozogamicin, which has been approved by the US Food and Drug Administration for treating adults with relapsed or refractory B-cell precursor ALL (BCP-ALL), couples the DNA double-strand break (DSB) inducer calicheamicin⁵  to an anti-CD22 antibody. Despite impressive complete remission (CR) rates and even minimal residual disease negativity, almost all patients in the INO-VATE ALL trial eventually relapsed without stem cell transplantation, demonstrating that inotuzumab ozogamicin monotherapy did not eradicate leukemia.² 

We recently demonstrated rapid induction of apoptosis in BCP-ALL by BH3 mimetics such as venetoclax.^6,7  Venetoclax directly releases apoptosis activators (mainly BIM) from BCL2⁷  linked to permeabilization of the mitochondrial outer membrane (MOM), the point of no return for mitochondrial apoptosis.⁸  Venetoclax synergizes with other MOM permeabilization (MOMP)-sensitizing drugs such as dexamethasone and dasatinib in BCR-ABL⁺ ALL^7,9,10  and proved curative in a xenograft BCR-ABL⁺ ALL mouse model.⁷ 

Primary samples were collected from patients who were newly diagnosed or had relapsed B-lineage ALL (see supplemental Table 1, available on the Blood Web site) who provided informed consent in accordance with the Declaration of Helsinki and the Ethics Committee of Hannover Medical School. All experimental procedures are described in the supplemental Materials.

We here hypothesized that induction of MOMP by venetoclax and DSB apoptosis signaling by calicheamicin may cooperate in BCP-ALL (Figure 1A). Induction of MOMP is linked to a decay of the mitochondrial membrane potential directly measureable in viable cells upon mitochondrial staining with tetramethylrhodamine ethyl ester (TMRE)⁷  (supplemental Figure 1A; supplemental Methods). Similarly, DSB-induced phosphorylation of the serine/threonine kinase ataxia telangiectasia mutated (ATM) on serine 1981 (pATM) and its target histone H2AX on serine 139 (γH2AX) (for review, see Blackford and Jackson¹¹ ) can be measured by flow cytometry¹²  (supplemental Figure 1B; supplemental Methods). We used BCP-ALL patient-derived xenograft (PDX) models (supplemental Table 1) and cell lines to analyze the induction of MOMP and the accumulation of pATM and γH2AX by venetoclax and inotuzumab ozogamicin, respectively. After 3 hours, venetoclax, but not inotuzumab ozogamicin or dexamethasone, decreased TMRE fluorescence (Figure 1B; supplemental Figure 1C). In contrast, only inotuzumab ozogamicin substantially induced pATM and γH2AX after 6 hours (Figure 1C-D; supplemental Figure 1D-E). No propidium iodide staining was detectable, but substantial caspase-3 cleavage was detectable in up to 15% of venetoclax-treated cells after 6 hours, which reflects the differential kinetics of activation (not execution) of apoptotic signaling by mitochondrial and pATM and γH2AX staining, respectively (supplemental Figure 1F-I). Dexamethasone did not directly induce MOMP or pATM or γH2AX at these early time points (Figure 1B-D; supplemental Figure 1C-E).

Next, sequential treatment was used to analyze interactions between direct induction of MOMP and DSB signaling (Figure 1E-F) in L707 PDX cells expandable on mesenchymal stem cells^13,14  and in 697 and BV173 cells. Cells were preincubated with venetoclax, dexamethasone, and inotuzumab ozogamicin for 24 hours; for the last 3 hours, venetoclax was added, and for the last 6 hours, inotuzumab ozogamicin was added. Venetoclax induced MOMP compared with untreated controls. Preincubation with inotuzumab ozogamicin and dexamethasone further enhanced this process (Figure 1E) and increased relative BIM loading to BCL2 (supplemental Figure 1J). In contrast, inotuzumab ozogamicin induced γH2AX expression compared with untreated controls, but γH2AX levels remained unchanged after preincubation with venetoclax or dexamethasone, which suggests that these drugs may not sensitize cells for DSB responses (Figure 1F). Immunoblot analyses demonstrated that inotuzumab ozogamicin induced expression of p53 and its targets BAX and PUMA (Figure 1G). These data are in line with directly induced MOMP apoptosis or DSB signaling at least partly converging on components of the mitochondrial apoptosis machinery.

We next analyzed venetoclax, inotuzumab ozogamicin, and dexamethasone alone and in fixed ratios¹⁵  in primary ALL and PDX samples in coculture with mesenchymal stem cells (supplemental Table 1).^7,14  Cell death was assessed by propidium iodide and human CD19 (huCD19) staining via flow cytometry after 48 hours. Drug responses were heterogeneous among ALL samples, and inotuzumab ozogamicin was effective in all but 1 sample (50% inhibitory concentration [IC₅₀] <10 nM Figure 1H). Venetoclax was effective in 7 of 11 samples, and dexamethasone was effective in 4 of 11 samples (IC₅₀ <1 µM). Next, combination therapies were analyzed using Chou-Talalay combination indices (CIs) with CI <1 indicative of drug synergy.¹⁵  The combination of venetoclax and inotuzumab ozogamicin reduced drug IC₅₀ values (supplemental Figure 1K) and behaved in a highly synergistic and additive manner (CI, 1; P020 cells) in all but 1 sample (Figure 1I), and the triple combination that included dexamethasone was synergistic in all samples (Figure 1J). No cytotoxicity was observed in normal peripheral blood mononuclear cells (supplemental Figure 1L).

On the basis of these data and previous data, combination therapy with venetoclax-dexamethasone can be more effective than venetoclax monotherapy (Scherr et al⁷ ; data not shown), so combination therapy was tested in vivo. We compared inotuzumab ozogamicin, venetoclax-dexamethasone, and the triple combination in Nod-Scid-IL2Rγnull (NSG) mice intravenously transplanted with 10⁶ P020 (hyperdiploid), P021(t(1;19)), and L707 (t(17;19)) cells and monitored noninvasively by in vivo bioluminescence imaging (BLI) based on luciferase reporter expression.¹⁶  These genetic changes are rare in adult ALL, but t(17;19), which is found mainly in adolescents, has an extremely poor outcome.¹⁷  Upon engraftment confirmed with BLI, mice were treated with vehicle control or with venetoclax-dexamethasone, inotuzumab ozogamicin, or the triple combination, and leukemic burden, leukemia-free survival (LFS), and overall survival were monitored. On the basis of the different inotuzumab ozogamicin IC₅₀’s, mice transplanted with the P020 and P021 models were treated with 10 µg/kg, whereas L707-engrafted mice were treated with 100 µg/kg, which is equivalent to human clinical dosing (supplemental Methods). In all 3 models, venetoclax-dexamethasone failed to induce CR with no survival benefit compared with controls (Figure 2A-D,G-H). In contrast, inotuzumab ozogamicin induced CR in 2 of 6 P021 mice but not in any P020 mice, despite a modest median survival benefit in both models (Figure 2A-D; data not shown). In contrast, triple combination therapy induced CR in all P020 and P021 mice and significantly prolonged median survival by 114 and 40 days, respectively. Dose dependency of inotuzumab ozogamicin therapy was next analyzed by treating P020 and P021 mice with 10-fold higher doses (100 µg/kg) (Figure 2E-F). All mice reached CR with inotuzumab ozogamicin monotherapy but eventually relapsed with a median survival of 140 days. In contrast, all mice survived BLI-negative upon treatment with the triple combination for up to 45 weeks after the start of therapy (Figure 2E-F). In the very-high-risk L707 model, the triple combination of inotuzumab ozogamicin and venetoclax-dexamethasone rapidly induced CR (no BLI signal was detectable) in all mice and prolonged median overall survival by 58 days, with 1 of 5 mice reaching long-term treatment-free LFS (Figure 2G-H). CD22 was expressed in all relapsed or refractory PDX samples analyzed, with expression levels depending on inotuzumab ozogamicin treatment in P021 cells, but no impairment of apoptosis initiated by inotuzumab ozogamicin and venetoclax was detectable (supplemental Figure 2A-G).

These data demonstrate the synergy of direct MOMP induction and DSB apoptosis signaling by concurrent treatment with venetoclax and inotuzumab ozogamicin in BCP-ALL. Indeed, only therapy with the combination of venetoclax-dexamethasone and inotuzumab ozogamicin induced CR in all 3 PDX models, with treatment-free long-term survival in all P020 and P021 mice being achieved in an inotuzumab ozogamicin dose-dependent manner. In contrast, dose-equivalent inotuzumab ozogamicin monotherapy failed to induce LFS corresponding to that in the INO-VATE ALL trial.²  Precise molecular mechanisms that mediate venetoclax and inotuzumab ozogamicin synergy and the contribution of dexamethasone to triple combination therapy remain to be determined. In addition, combined treatment may target different survival signals or threshold levels for the induction of apoptosis essential for ALL cells in vivo. Combination therapy with venetoclax, inotuzumab ozogamicin, and steroids should be evaluated in clinical trials of high-risk BCP-ALL patients either to optimize definitive pharmacotherapy or to deepen remission of leukemia before consolidation with cellular therapy.

The authors thank Andrea Hoffmann (Department of Orthopedic Surgery, Hannover Medical School) for providing mesenchymal stem cells and Jonathan Lühmann for conducting the optical genome mapping for retrospective cytogenetic analyses. The authors acknowledge Iris Dallmann for excellent technical help and the staff of the Central Animal Facility and Matthias Ballmaier from the Cell Sorting Core Facility (supported, in part, by the Braukmann-Wittenberg-Herz-Stiftung and the Deutsche Forschungsgemeinschaft) of Hannover Medical School.

This work was supported by H.W. and J. Hector-Stiftung und Stiftung Gerdes, and by a Cancer Research UK program grant (C27943/A12788) (O.H.).

Contribution: H.K., M.E., and M.S. conceived the study and designed the experiments; H.K., K.B., U.K., C.S., S.E., S.R.T., and D.S. performed experiments and analyzed and interpreted the data; O.H., M.H., D.H.-K., and A.G. provided technical and material support; H.K., M.E., M.S., and O.H. wrote the manuscript; and all authors reviewed the manuscript before submission.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Matthias Eder, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; e-mail: eder.matthias@mh-hannover.de; and Michaela Scherr, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; e-mail: scherr.michaela@mh-hannover.de.