Targeting Mcl-1 by AMG-176 During Ibrutinib and Venetoclax Therapy in Chronic Lymphocytic Leukemia

Abstract

B-cell receptor (BCR) signaling pathway and Bcl-2 family prosurvival proteins, specifically Bcl-2 and Mcl-1, are functional in the pathobiology of chronic lymphocytic leukemia (CLL). A pivotal and apical molecule in the BCR pathway is Bruton’s tyrosine kinase (BTK). Together, BTK, Bcl-2, and Mcl-1 participate in the maintenance, migration, proliferation, and survival of CLL cells. Several ongoing and published clinical trials in CLL reported high rates of remission, namely, undetectable measurable residual disease (u-MRD) status with combined BTK inhibitor ibrutinib and Bcl-2 antagonist, venetoclax. While the majority of patients achieve complete remission with undetectable-measurable residual disease, at least one third of patients do not achieve this milestone. We hypothesized that cells persistent during ibrutinib and venetoclax therapy may be sensitive to combined venetoclax and Mcl-1 inhibitor, AMG-176. To test this hypothesis, we took peripheral blood samples at baseline, after Cycle 1 and Cycle 3 of ibrutinib monotherapy, after one week and 1 cycle of ibrutinib plus venetoclax therapy. These serial samples were tested for pharmacodynamic changes and treated in vitro with AMG-176 or in combination with venetoclax. Compared to C1D1 cells, residual cells during ibrutinib and venetoclax treatment were inherently resistant to endogenous cell death. Single agent exposure induced some apoptosis but combination of 100 nM venetoclax and 100 or 300 nM of AMG-176 resulted in 40–100% cell death in baseline samples. Cells obtained after four cycles of ibrutinib and one cycle of venetoclax, when treated with such concentration of venetoclax and AMG-176, showed 10–80% cell death. BCR signaling pathway, measured as autophosphorylation of BTK was inhibited throughout therapy in all post-therapy samples. Among four anti-apoptotic proteins, Mcl-1 and Bfl-1 decreased during therapy in most samples. Proapoptotic proteins decreased during therapy. Collectively, these data provide a rationale to test Mcl-1 antagonists alone or in combination in CLL during treatment with ibrutinib and venetoclax.

Background

Survival, proliferation, migration, and maintenance of normal B cells and chronic lymphocytic leukemia (CLL) B cells are dependent on B-cell receptor (BCR) signaling pathway (1, 2). A pivotal molecule in the BCR signaling pathway is Bruton’s tyrosine kinase (BTK) which is at the apex of this nexus. Ibrutinib irreversibly inhibits BTK by covalently binding to the C481 residue of the kinase domain (3). The importance of BTK inhibition was clearly demonstrated by achievement of long-term progression-free and overall survival with ibrutinib monotherapy in patients with CLL versus standard chemotherapy or chemoimmunotherapy (4, 5). As a consequence, the drug was FDA approved as monotherapy for all CLL subgroups, namely, elderly patients (6) and patients with 17pdel (7). However, the complete remission rate was less than 5% in previously untreated patients with CLL (5).

In addition to the importance of BCR signaling pathway in the pathophysiology of CLL disease, Bcl-2 family prosurvival proteins (8, 9) were established as primary molecules responsible for apoptosis evasion of the malignant B cells. Among several genetic alterations in CLL, the most prevalent is deletion of chromosome 13q which occurs in 2/3 of patients (10). Identification of microRNA in this region and their role in overexpression of Bcl-2 and Mcl-1 further established role of these two antiapoptotic proteins in the biology of CLL (11, 12). Investigations of protein–protein interaction and fraction-based structural activity relationship for drug design were primary reasons for creation of navitoclax followed by venetoclax, a drug that exclusively neutralizes Bcl-2 antiapoptotic protein (13, 14). Its success as single-agent further established importance of Bcl-2 in the pathogenesis of CLL and especially in survival of malignant lymphocytes in patients. Venetoclax monotherapy results in 20% complete remissions and 5% undetectable measurable residual disease (u-MRD) status (15, 16). Hence, this remarkable pharmacological agent was also identified to be best used in mechanism-based combinations (17–19).

Both ibrutinib and venetoclax are approved, established, and effective single agents for treatment of CLL and other B-cell malignancies such as mantle cell lymphoma. However, in each case responses are mostly partial remissions; only a minority of patients achieve complete remissions with single agent treatments. Preclinically, we and others showed that levels of Mcl-1 (20) and Mcl-1 and BCL-xl (21) decreased after ibrutinib therapy resulting in priming of these cells for venetoclax-induced cytotoxicity. This decrease in survival proteins may be due to inhibition of BCR pathway and disruption of chemokine-controlled integrin-mediated homing of CLL cells (22). Murine models further provided evidence for benefits of combining BTK inhibitors with venetoclax (23, 24). Clinically, these drugs have non-overlapping toxicities, complementary yet distinct targets in CLL cells, and collective impact on CLL cells in all three niches such as blood, bone marrow, and lymph nodes (19). Ex vivo model that promotes CLL cell proliferation demonstrated that each drug works on different subpopulation resulting in synergy (25). Both drugs have been used in combination during clinical trials for B-cell malignancies such as mantle-cell lymphoma (MCL) (26), treatment-naïve CLL (18, 27, 28) or previously treated CLL (29, 30). Additional studies are further establishing clinical utility of the combination for CLL cells, namely, defined duration of therapy after achievement of u-MRD status (31, 32). Finally, results of the recent randomized GLOW trial demonstrated superior efficacy and safety of combined ibrutinib and venetoclax over chemoimmunotherapy (33). Pharmacodynamic endpoints during ibrutinib and venetoclax combination trials suggest 50–70% complete remission and u-MRD status in bone marrow or peripheral blood. Since all patients do not achieve CR or u-MRD status, these data suggest that there is/are additional molecules that provide survival advantage to CLL cells.

Mcl-1 was recognized among the top molecule in cancers for somatic copy number alterations (34). Mcl-1 protein levels have been shown to be important in the survival of CLL cells (20, 21, 35–41). Recently, direct inhibitors of Mcl-1 have been designed by Astra Zeneca [AZD-5991 (42)], Amgen [AMG-176 (43, 44)] and Novartis [S63845 and S64315 (45, 46)]. Clinical trials (NCT03218683; NCT02675452; NCT02992483) in liquid tumors are ongoing with these agents. Previously we established in preclinical studies that single-agent AMG-176 and S63845 were effective at inducing apoptosis in CLL cells (44, 47).

Based on this background, we hypothesized that AMG-176 will induce apoptosis in CLL cells previously treated with ibrutinib or combined ibrutinib and venetoclax therapy. To test this postulate, we took peripheral blood samples at baseline, after Cycle 1 and Cycle 3 of ibrutinib monotherapy, after one week of combination ibrutinib + venetoclax therapy, and after 1 complete cycle of combined ibrutinib and venetoclax. These serial samples were tested for pharmacodynamic changes and treated in vitro with AMG-176.

Methods

Clinical Trial

The associated clinical trial was an investigator-initiated, open-label, phase II study of ibrutinib and venetoclax combination for patients with high-risk treatment-naive CLL or relapsed/refractory CLL (ClinicalTrials.gov number, NCT02756897). Patients initiated treatment with oral ibrutinib (420 mg once daily) as a single-agent for the first 3 cycles. The goals of ibrutinib monotherapy were to mobilize cells from lymph nodes, reduce tumor burden and consequently decrease the risk of tumor lysis syndrome (TLS), and to have CLL cells with lower level of Mcl-1 protein. After the 3 cycles of ibrutinib, venetoclax was initiated at the start of cycle 4 (Figure 1A). Oral venetoclax was dose-escalated in a weekly fashion (20 mg → 50 mg → 100 mg → 200 mg → 400 mg) to a target dose of 400 mg once daily, according to the prescribing information for venetoclax in CLL. Each cycle is four weeks. Combined venetoclax and ibrutinib were administered for a total of 24 cycles. Additional information regarding long-term treatment is provided in the original publication and in the recent update of the clinical trial results (18, 27).

Figure 1

Patient Population

All patients in the study had an iwCLL treatment indication and were enrolled at The University of Texas MD Anderson Cancer Center (MDACC), Houston, TX, USA. Eligibility criteria were previously described in detail (18). Patients previously treated with ibrutinib or venetoclax were excluded. All patients were assessed for IGHV mutation status, chromosome abnormalities by fluorescence-in-situ-hybridization (FISH), and conventional cytogenetics. Targeted sequencing of 29 genes, including TP53, SF3B1, NOTCH1, BIRC3, ATM, was performed on tumor DNA from the pre-treatment BM aspirate.

Patient Sample Collection and Processing

For pharmacodynamic investigations and ex vivo incubations with AMG-176 and/or venetoclax, serial peripheral blood samples were collected from patients during therapy. Peripheral blood was obtained from patients with CLL who provided written informed consent as part of a protocol approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center in accordance with the Declaration of Helsinki. Blood samples were obtained at different days (D) and cycles (C). Time points included pre-treatment (C1D1), after one cycle of ibrutinib monotherapy (C2D1), after 3 cycles of ibrutinib monotherapy (C4D1), after a week of combination ibrutinib + venetoclax therapy (C4D8), and after one cycle of the combination therapy (C5D1). Altogether, ~100 blood samples were obtained from 20 patients to be used for different experimental purposes. Baseline clinical and molecular characteristics of these patients are summarized in Table 1 and described in the Results section.

In all cases, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density centrifugation (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) and suspended in Roswell Park Memorial Institute 1640 (RPMI 1640) media supplemented with 10% human serum (Sigma-Aldrich, St. Louis, MO). These samples were always used fresh for each experiment.

Drugs

AMG-176 and venetoclax were purchased from Chemietek (Indianapolis, IN) and Xcessbio Company (San Diego, CA), respectively. For both drugs, stock solutions were made in dimethyl sulfoxide (DMSO; Sigma-Aldrich). All incubations were for 24 h at the indicated drug concentration alone or in combination. DMSO only was used as a vehicle control.

Ex Vivo Drug Incubations

CLL cells were isolated from the peripheral blood samples at 5 different time points and incubated ex vivo with vehicle (DMSO) only (Figure 1); AMG-176 at different concentrations (Figure 2); 100 nM AMG-176, 300 nM AMG-176, 100 nM venetoclax only; or combination of 100 nM venetoclax with 100 or 300 nM AMG-176 (Figures 3–6). All incubations were for 24 h.

Figure 2

Figure 3

Cytotoxicity Assay

Cells were treated either with DMSO (vehicle control), or with single or combined drugs as indicated above for 24 h. For apoptosis assay, cells were stained with annexin V and propidium iodide (PI). Stained cells were analyzed by flow cytometry (BD Accuri C6, BD Biosciences). To calculate cell death, values from early apoptotic (Annexin⁺ PI⁻), late apoptotic (Annexin⁺ PI⁺) and necrotic (Annexin⁻ PI⁺) cells were included and labeled as % Annexin V/PI positive. Cells that were negative for both stains were considered live cells. Cell death of vehicle-treated samples was subtracted from drug-treated samples.

Immunoblot Analyses

CLL cell pellets were suspended in modified RIPA buffer, with protease and phosphatase inhibitors, to extract total proteins. Whole cell lysates (20–30 µg) were used to perform the immunoblots and were run on Criterion protein gels (4–12% gradient gels; Bio-Rad). The gels were transferred onto Bio Rad immune-blot polyvinylidene difluoride (PVDF) membrane (Bio Rad #1620177), cut into slices, and incubated with specific antibodies, followed by visualization with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Antibodies for specific proteins are listed in Supplementary Table 1.

Quantitation of Immunoblots

The signal intensity of each band of target protein and also internal control (either Vinculin or GAPDH) was measured using Licor Image Studio software following manufacturer’s guideline (www.Licor.com). Values were normalized using loading control and then were compared to that in vehicle control. Final data were plotted into GraphPad Prism 7 for representation.

Statistical Analysis

All graphs were created using Prism software (GraphPad). To determine statistical significance, Prism software was used and samples in the control (DMSO treated) were compared with drug treated cells using one-tailed paired Student’s t-test. P-values lower than 0.05 were considered significant. Values are provided either in the figure legend, included in the figure as a table, or included in the figure as an asterisk.

Results

Patient Characteristics

Sample collection, drug doses, and protocol schema are provided (Figure 1A). Characteristics of patients (n = 20) is described (Table 1). Majority of patients were treatment naïve. All except 2 patients had unmutated IGHV. ZAP-70 status, B2M level, hematological data, and CLL disease staging are listed in Table 1. All patients had at least one of the five feature: del(17p), mutated TP53, del(11q), unmutated IGHV, or age ≥65 years. Unique patient identifiers are provided in the first column and these numbers are listed in the figures.

Inherent Capability of Cell Death

During therapy, WBC count in peripheral blood decreased after ibrutinib monotherapy and, in particular, after the addition of venetoclax (Figure 1B). Lymphocyte count in the peripheral blood was similar for the first four time points but decreased at C5D1 which likely reflects venetoclax-induced apoptosis (Figure 1C). When grown in culture in regular medium (containing 10% human serum), CLL lymphocytes undergo cell death in a time-dependent manner. This endogenous cell death is heterogeneous among patients. To determine if inherent cell death would be different after treatment with ibrutinib or ibrutinib plus venetoclax, pretreatment and post-therapy samples were cultured in DMSO (vehicle control) for 24 h. Baseline (pretreatment) sample showed variation in endogenous cell death ranging from 0 to 55%. Compared to this baseline (C1D1) sample, paired samples at different time points showed much less cell death (Figure 1D). The values were significantly different in paired samples at every time point starting with C2D1 and ending with C5D1 (p = 0.04 to 0.002; Figure 1E). These data suggest that persister cells in the peripheral blood after treatment with ibrutinib alone or in combination with venetoclax were relatively resistant to inherent cell death.

Sensitivity of CLL Lymphocytes to AMG-176 During Ibrutinib Monotherapy and Ibrutinib Plus Venetoclax Therapy

To select a dose of AMG-176 and to test if CLL lymphocytes show more or less sensitivity to AMG-176 after treatment with ibrutinib or combination ibrutinib/venetoclax, four patient samples (#518, 356, 214, 221) were used for this investigation; each patient is represented by a color which is defined in the figure legend (Figure 2). Similar to endogenous cell death data, there was heterogeneity for cell death among baseline (C1D1) samples, yet there was evidence of some dose-dependency. However, in most cases, the extent of cell death with exogenous AMG-176 decreased after treatment with ibrutinib or combination (C4D8 and C5D1). These data suggest that not only do lymphocytes show relatively less sensitivity to endogenous cell death, they were less sensitive to AMG-176-induced cell death also after ibrutinib or ibrutinib plus venetoclax therapy. Based on dose-dependency (Figure 2) and achievable plasma concentrations, we selected 100 and 300 nM AMG-176 for future combination experiments presented in Figure 3.

Sensitivity of CLL Lymphocytes to AMG-176, Venetoclax, or Their Combination During Ibrutinib Monotherapy and Ibrutinib Plus Venetoclax Therapy

We evaluated if peripheral blood cells at baseline (C1D1), during ibrutinib alone (C2D1, C4D1), or ibrutinib and venetoclax (C4D8 and C5D1) were sensitive to additional ex vivo incubations with 100 nM and 300 nM AMG-176, 100 nM venetoclax, or venetoclax in combination with 100 nM or 300 nM AMG-176 (Supplementary Figure 1 and Table 2). Cell death data from 10 to 15 patients (numbers under abscissa) are presented in separate graphs for each time point (Figures 3A–E) and data for each drug alone and in combinations were compared with DMSO for each time point with statistical analyses or venetoclax alone was compared with combination (Figure 3F).

Prior to any therapy (i.e., C1D1 time point) cells were sensitive to AMG-176 alone (Mean 26% and 42% cell death, p = 0.026 and <0.0001 at 100 and 300 nM, respectively), venetoclax alone (Mean 61% cell death, p = 0.0002) and combination resulted in similar cell death as venetoclax alone (mean 70% cell death with p = <0.001 at both AMG-176 concentrations) (Figures 3A, 3F and Table 2).

Cells from patients receiving ibrutinib monotherapy (C2D1 and C4D1) showed a mean 64.5% ± 7.8% and 54.1% ± 7.2% venetoclax-induced cell death (p = <0.0001 at both time points). Moderate yet significant increase in cell death was demonstrated when 100 nM (70.6% ± 5.8% and 67.1% ± 6.9% cell death; p = 0.04 in C2D1 and 0.002 in C4D1 sample) or 300 nM AMG-176 (70.2% ± 6.1% and 70.6% ± 6.3%; p = 0.04 in C2D1 and 0.002 in C4D1 sample) was added exogenously (Figures 3B, C, F, Table 2).

Cells taken from patients after 3 cycles of ibrutinib and one week of ibrutinib plus venetoclax (at 20 mg daily dose; C4D8; Figure 1A) showed less cell death compared to C2D1 and C4D1 samples (ibrutinib monotherapy) with ex vivo incubations with each drug alone or in combination (Table 2). However, compared to venetoclax alone (45.3% ± 6.3%), these cells had higher level of apoptosis when both drugs (AMG-176 and venetoclax) were added exogenously (59.9% ± 6.3% p = 0.002 at 100 nM and 64.4% ± 6.2%, p = 0.003 at 300 nM AMG-176) (Figure 3D and Table 2). Similarly, at C5D1 time point (with 4 cycles of ibrutinib and with 4 weeks of ramp-up dosing of venetoclax; Figure 1A), compared to venetoclax alone (19.7% ± 3.5%), the combination of venetoclax and 100 nM AMG-176 (36.8% ± 4.9%, p ≤0.0002) or 300 nM AMG-176 (42.5% ± 6.2%, p = <0.0002) resulted in increased cell death (Figure 3E and Table 2).

Inhibition of the BCR Pathway Signaling During Ibrutinib Monotherapy and Ibrutinib Plus Venetoclax Therapy

The action of ibrutinib is on phosphorylation of BTK and, by extension, inhibition of the BCR signaling pathway while venetoclax targets Bcl-2, one of the most abundant antiapoptotic proteins in CLL lymphocytes. Hence, in 16 patient samples where enough cells were available, we performed immunoblot assay for BCR transduction proteins and Bcl-2 pro- and anti-apoptotic proteins (Supplementary Figure 2). The protein levels were quantitated, normalized with loading control, and expressed as percent of baseline (Supplemental Figure 3, 4A–C). Among the BCR signaling pathway proteins, BTK and PLCγ2 were evaluated; statistical values are provided in the figure legend (Figure 4). Decrease in phospho-BTK levels was observed starting with C2D1 and further declined with additional treatment (Figure 4A). For phospho-PLCγ2, there was a trend in a mean decrease in the value, but this was only significantly different for C4D8 (Figure 4B). Median value of phospho-PLCγ2 at Tyr1217 remained similar at different time points and was not significantly different (Supplementary Figure 5); of note, this site is not phosphorylated by BTK. Previously we showed that total BTK transcript and total protein levels decrease in CLL cells during ibrutinib therapy (48). This was also observed in the current therapy and was significantly different at all time points compared to pretreatment value (Figure 4C). Similarly, significant decline in total PLCγ2 was observed at all time points compared to baseline value (Figure 4D).

Figure 4

Changes in Bcl-2 Antiapoptotic Family Proteins During Ibrutinib Monotherapy and Ibrutinib Plus Venetoclax Therapy

Four of the 6 members of the Bcl-2 family of anti-apoptotic proteins were detected in CLL lymphocytes (Supplementary Figures 3,4 and Figures 5A–D). Bcl-2 protein showed heterogeneous response among patients treated with ibrutinib which was not significantly different than the baseline value. However, after addition of venetoclax, the levels of Bcl-2 proteins (Figure 5A) were significantly different at C4D8 (p = 0.0095) and C5D1 (0.0160). Bcl-XL protein levels (Figure 5B) were not significantly changed, although there was a trend for an increase in some patient samples (C5D1; p = 0.0463). In contrast to Bcl-2 and Bcl-XL, compared to C1D1 values, aggregate values for Mcl-1 and Bfl-1 proteins (Figures 5C, D) continuously and significantly decreased at all four time points (p = 0.0103 − <0.0001). However, regarding Mcl-1, there is heterogeneity among 16 patient samples (Supplementary Figure 6). For the 4 patients, who did not achieve u-MRD, Mcl-1 levels remained unchanged in patient 214; decreased precipitously in patient 888; decreased first and then increased in patients 098 and 614. CLL cells after ibrutinib treatment had heterogeneous response to uncleaved PARP protein, which was not statistically significantly different, however, after addition of venetoclax (C4D8 and C5D1), there was a significant decline in PARP protein indicating induction of apoptosis after addition of venetoclax (Supplementary Figure 7).

Figure 5

Changes in Bcl-2 Proapoptotic Family Proteins During Ibrutinib Monotherapy and Ibrutinib Plus Venetoclax Therapy

There are several members of the Bcl-2 family proapoptotic proteins. Six of these proteins were tested in CLL lymphocytes (Supplementary Figure 4). Bax and Bak are multidomain proapoptotic Bcl-2 family proteins needed for intrinsic cell death (49). Levels of Bak showed some perturbations but generally the changes were not significant (p-value, 0.05–0.23). In contrast, level of Bax protein showed a 50% decline which was consistent in all 4 time points after start of ibrutinib and ibrutinib plus venetoclax therapy (Figures 6A, B). Four different BH3-only domain Bcl-2 family proteins were also tested. Bcl-2 Rambo levels decreased in all patients (p ≤0.0004), however, the extent was heterogeneous among patients (Figure 6C). Bim protein (Figure 6D) had heterogeneous and non-significant (p = 0.123) response after one cycle of ibrutinib but then declined at other time points (p = 0.011 − <0.0001). Compared to baseline value in C1D1 samples, both Noxa and Puma proteins (Figures 6E, F) were significantly reduced after start of ibrutinib and decreased further after addition of venetoclax (p = 0.03 − <0.0001).

Figure 6

Discussion

Our current investigations suggest that peripheral blood cells that persist after a few cycles of ibrutinib and one cycle of venetoclax therapy are inherently less primed for apoptosis. While they do not show high sensitivity to either AMG-176 or venetoclax alone, they are sensitized ex vivo to treatment with combined Mcl-1 and Bcl-2 antagonists, AMG-176 and venetoclax.

Pharmacodynamic investigations during ibrutinib monotherapy showed decline in Mcl-1 protein levels with no or limited changes in the level of Bcl-2 protein (20). We observed similar changes in the level of Mcl-1 protein with another covalent BTK inhibitor, acalabrutinib (50, 51) and was also reported by another group (24) indicating the role of the BCR signaling pathway in this pharmacodynamic effect. Consistent with this postulate, PI3 kinase inhibitors such as idelalisib (52) and duvelisib (53) incubations or therapy resulted in modulation of Mcl-1 protein levels. Our present investigations in ibrutinib followed by ibrutinib and venetoclax couplet clinical trial patient samples further establish significant reduction in Mcl-1 protein (Figure 5C). Previously we have demonstrated in CLL cells that AMG-176-mediated CLL cell death was inversely proportional to level of Mcl-1 protein (44). The relevant and unanswered question with this regard is whether the Mcl-1 protein can be targeted by direct antagonist such as AMG-176 to sensitize CLL cells that remain during treatment with ibrutinib and venetoclax.

AMG-176 is a potent antagonist of Mcl-1; in cell free system the Ki value is <1 nM. In whole cells, the drug was tested in different leukemias, lymphoma, and multiple myeloma cell lines (43). Pertaining to current work, AMG-176 was tested in primary CLL cells from 74 patient samples showing cell death when 300 nM concentration was used (44). We also observed an inverse correlation of AMG-176 induced cell death and endogenous levels of Mcl-1. Considering this relationship, ibrutinib-mediated decrease in Mcl-1 provides a rationale to combine AMG-176 during or after combined treatment with ibrutinib and venetoclax.

The primary target of ibrutinib is BTK which is critical in the BCR signaling pathway. Its centrality in the BCR signaling pathway and maintenance of B-cells became clear with the therapeutic success of BTKi (54). Pharmacodynamic analysis of BCR signaling using phospho-BTK protein as a biomarker clearly demonstrated that in circulating CLL cells of the 16 patients evaluated, this pathway was hampered during the four cycles of ibrutinib monotherapy. In addition to a decrease in phospho-BTK, total BTK protein (Figures 4A, B) was also diminished which is consistent with our prior observations (48, 55).

The current project did not focus on mechanism of Mcl-1 decline after ibrutinib therapy and collateral induction of apoptosis. Somatic copy number alteration studies in many cancers identified Mcl-1 as the topmost genes with increased copy number (34). It is also highly expressed in many human cancers (56). Specific to CLL, this prosurvival protein was shown to be essential during early lymphoid development. Furthermore, maintenance of mature T and B-lymphocytes is dependent upon this protein (57). These observations underscore centrality of this protein in lymphocytes and suggest demise of these cells when Mcl-1 dissipates. However, it is not clear how this protein declines with ibrutinib therapy. Activation of the BCR signaling pathway leads to activation of several transcription factors, which may be responsible for transcription of MCL-1 gene. Further, Mcl-1 is a short-lived protein and diminishes with a half-life of <1 h (58). Additionally, onset of apoptosis of leukemia cells can further degrade Mcl-1 as this protein has two caspase cleavage sites. Once cleaved, the C-terminal domain of Mcl-1 acts as a pro-apoptotic molecule after caspase-3 cleavage which pushes intrinsic apoptosis (59). Caspase-mediated cleavage and dissipation of Mcl-1 may be a highly likely scenario in CLL cells from patients treated with ibrutinib and venetoclax therapy, however, such postulates need to be tested.

Bcl-2, Mcl-1, Bfl-1, and Bcl-XL are four primary antiapoptotic molecules of the Bcl-2 family survival proteins that are expressed in CLL lymphocytes. Their abundance results in evasion of apoptosis in the malignant lymphocytes. While Bcl-2 and Mcl-1 are being antagonized with our current approach in the present paper, Bfl-1 and Bcl-XL remain untargeted. Bfl-1 protein, however, was consistently and continuously decreased during therapy while Bcl-XL levels were significantly induced, especially at the C5D1 time point (Figure 5B). Further, there appears to be heterogeneity and 50% of the samples showed a high increase in Bcl-XL. It is possible that circulating CLL cells with high level of Bcl-XL are being enriched after in vivo treatment with ibrutinib and venetoclax. On the other hand, mimicking in vivo environment during in vitro investigations in CLL and mantle cell lymphoma cells also showed Mcl-1, Bcl-XL, and survivin as primary determinants of ibrutinib and venetoclax resistance (41). Collectively, these data also provide rationale to test Bcl-XL inhibitors after combined BTKi and venetoclax.

Several investigator-initiated trials and multicenter studies have now established clinical success of ibrutinib and venetoclax combination in CLL and also in mantle cell lymphoma (26). One of the first trials of this couplet in CLL was conducted at our center and reported high rates of complete remission, undetectable measurable residual disease (u-MRD), overall survival and progression-free survival in previously untreated patients with poor prognosis attributes (18). Impressively, high remission, survival, and u-MRD status were maintained even after three years of therapy which included one year without treatment in patients who had u-MRD or with ibrutinib maintenance therapy for patients who had detectable measurable disease (27). CAPTIVATE study further attested superiority of this combination resulting in the 30-month progression free survival rate of ~95% across all treated patients. Similar data are emerging from many different institutes in large studies (28, 29) and also for patients with relapsed/refractory CLL (29, 30, 60). Recent results from the randomized trial of ibrutinib and venetoclax versus chlorambucil and obinutuzumab (CLL GLOW trial) validated the ibrutinib and venetoclax couplet therapy for CLL as a time-limited oral targeted therapy for patients with CLL (33). Collectively, these data demonstrate high u-MRD in both untreated and previously treated CLL but also emphasize that there are patients who do not respond to this combination, underscoring a need for next step. Our current data clearly provide evidence in primary target cells during the ibrutinib and venetoclax couplet therapy. Our pharmacokinetic profiling and pharmacodynamic investigations demonstrate Mcl-1 inhibitors such as AMG-176 as a suitable partner with ibrutinib and venetoclax.

All three direct inhibitors of Mcl-1, e.g. AMG-176, have been tested in the clinic for hematological malignancies. However, untoward toxicities of these agents have hampered the enthusiasm. Modification of these drugs and structure activity relationship may result in a better clinical candidate. Additionally, new schedules are being tested in the clinic trials which may help ameliorate toxicity. An indirect approach to target Mcl-1 is to transiently inhibit transcription or protein translation of this anti-apoptotic protein using transcription and translation inhibitors. Mcl-1 has short transcript turn-over and short protein half-life suggesting transcription and translation inhibitors as an option to reduce levels of Mcl-1. CDK9 and CDK7 are key kinases that phosphorylate serine residues on the heptamer repeats on the C-terminal of RNA polymerase II which is critical for mRNA syntheses (61). Flavopiridol and SNS-032 are potent inhibitors of CDK7 and 9; treatment with these agents decreased levels of short-lived transcripts such as Mcl-1 and Myc. Such strategies have been successful both during in vitro incubations and during therapy for CLL (62, 63), and Mcl-1 was predictor of cell death (64). Because Mcl-1 protein half-life is short, inhibition of translation could also play a role in dissipation of Mcl-1 protein and induction of apoptosis. Clinical candidates such as omacetaxine and silvestrol, both natural products, block protein synthesis and were utilized as protein translation inhibitor (65, 66). These transcription and translation inhibitors need to be tested on persister cells after combined ibrutinib and venetoclax therapy.

Taken together, our data provide evidence for additional combination strategies with ibrutinib and venetoclax for patients with CLL. Because direct and indirect antagonists of Mcl-1 are available for clinical use, such approach could be translated to clinic for patients with B-cell malignancies in general and CLL in particular.

Funding

This work was supported in part by a CLL Global Research Foundation Alliance grant and MD Anderson’s CLL Moon Shot™ program. XY received scholarship from China to work as a visiting scientist.

Publisher’s Note

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References

• 1

  KippsTJStevensonFKWuCJCroceCMPackhamGWierdaWGet al. Chronic Lymphocytic Leukaemia. Nat Rev Dis Primers (2017) 3:16096. doi: 10.1038/nrdp.2017.8

  • CrossRef
  • Google Scholar

• 2

  StevensonFKForconiFKippsTJ. Exploring the Pathways to Chronic Lymphocytic Leukemia. Blood (2021) 138(10):827–35. doi: 10.1182/blood.2020010029

  • CrossRef
  • Google Scholar

• 3

  HonigbergLASmithAMSirisawadMVernerELouryDChangBet al. The Bruton Tyrosine Kinase Inhibitor PCI-32765 Blocks B-Cell Activation and is Efficacious in Models of Autoimmune Disease and B-Cell Malignancy. Proc Natl Acad Sci (2010) 107(29):13075. doi: 10.1073/pnas.1004594107

  • CrossRef
  • Google Scholar

• 4

  ByrdJCBrownJRO’BrienSBarrientosJCKayNEReddyNMet al. Ibrutinib Versus Ofatumumab in Previously Treated Chronic Lymphoid Leukemia. N Engl J Med (2014) 371(3):213–23. doi: 10.1056/NEJMoa1400376

  • CrossRef
  • Google Scholar

• 5

  BurgerJATedeschiABarrPMRobakTOwenCGhiaPet al. Ibrutinib as Initial Therapy for Patients With Chronic Lymphocytic Leukemia. N Engl J Med (2015) 373(25):2425–37. doi: 10.1056/NEJMoa1509388

  • CrossRef
  • Google Scholar

• 6

  O’BrienSFurmanRRCoutreSESharmanJPBurgerJABlumKAet al. Ibrutinib as Initial Therapy for Elderly Patients With Chronic Lymphocytic Leukaemia or Small Lymphocytic Lymphoma: An Open-Label, Multicentre, Phase 1b/2 Trial. Lancet Oncol (2014) 15(1):48–58. doi: 10.1016/S1470-2045(13)70513-8

  • CrossRef
  • Google Scholar

• 7

  O’BrienSJonesJACoutreSEMatoARHillmenPTamCet al. Ibrutinib for Patients With Relapsed or Refractory Chronic Lymphocytic Leukaemia With 17p Deletion (RESONATE-17): A Phase 2, Open-Label, Multicentre Study. Lancet Oncol (2016) 17(10):1409–18. doi: 10.1016/S1470-2045(16)30212-1

  • CrossRef
  • Google Scholar

• 8

  BirkinshawRWCzabotarPE. The BCL-2 Family of Proteins and Mitochondrial Outer Membrane Permeabilisation. Semin Cell Dev Biol (2017) 72:152–62. doi: 10.1016/j.semcdb.2017.04.001

  • CrossRef
  • Google Scholar

• 9

  HotchkissRSStrasserAMcDunnJESwansonPE. Cell Death. N Engl J Med (2009) 361(16):1570–83. doi: 10.1056/NEJMra0901217

  • CrossRef
  • Google Scholar

• 10

  CalinGADumitruCDShimizuMBichiRZupoSNochEet al. Frequent Deletions and Down-Regulation of Micro- RNA Genes Mir15 and Mir16 at 13q14 in Chronic Lymphocytic Leukemia. Proc Natl Acad Sci USA (2002) 99(24):15524–9. doi: 10.1073/pnas.242606799

  • CrossRef
  • Google Scholar

• 11

  CimminoACalinGAFabbriMIorioMVFerracinMShimizuMet al. miR-15 and miR-16 Induce Apoptosis by Targeting BCL2. Proc Natl Acad Sci USA (2005) 102(39):13944–9. doi: 10.1073/pnas.0506654102

  • CrossRef
  • Google Scholar

• 12

  PekarskyYBalattiVCroceCM. BCL2 and miR-15/16: From Gene Discovery to Treatment. Cell Death Differ (2018) 25(1):21–6. doi: 10.1038/cdd.2017.159

  • CrossRef
  • Google Scholar

• 13

  OltersdorfTElmoreSWShoemakerARArmstrongRCAugeriDJBelliBAet al. An Inhibitor of Bcl-2 Family Proteins Induces Regression of Solid Tumours. Nature (2005) 435(7042):677–81. doi: 10.1038/nature03579

  • CrossRef
  • Google Scholar

• 14

  SouersAJLeversonJDBoghaertERAcklerSLCatronNDChenJet al. ABT-199, a Potent and Selective BCL-2 Inhibitor, Achieves Antitumor Activity While Sparing Platelets. Nat Med (2013) 19(2):202–8. doi: 10.1038/nm.3048

  • CrossRef
  • Google Scholar

• 15

  RobertsAWDavidsMSPagelJMKahlBSPuvvadaSDGerecitanoJFet al. Targeting BCL2 With Venetoclax in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med (2015) 374(4):311–22. doi: 10.1056/NEJMoa1513257

  • CrossRef
  • Google Scholar

• 16

  StilgenbauerSEichhorstBScheteligJCoutreSSeymourJFMunirTet al. Venetoclax in Relapsed or Refractory Chronic Lymphocytic Leukaemia With 17p Deletion: A Multicentre, Open-Label, Phase 2 Study. Lancet Oncol (2016) 17(6):768–78. doi: 10.1016/S1470-2045(16)30019-5

  • CrossRef
  • Google Scholar

• 17

  FischerKAl-SawafOBahloJFinkA-MTandonMDixonMet al. Venetoclax and Obinutuzumab in Patients With CLL and Coexisting Conditions. N Engl J Med (2019) 380(23):2225–36. doi: 10.1056/NEJMoa1815281

  • CrossRef
  • Google Scholar

• 18

  JainNKeatingMThompsonPFerrajoliABurgerJBorthakurGet al. Ibrutinib and Venetoclax for First-Line Treatment of CLL. N Engl J Med (2019) 380(22):2095–103. doi: 10.1056/NEJMoa1900574

  • CrossRef
  • Google Scholar

• 19

  TimofeevaNGandhiV. Ibrutinib Combinations in CLL Therapy: Scientific Rationale and Clinical Results. Blood Cancer J (2021) 11(4):1–12. doi: 10.1038/s41408-021-00467-7

  • CrossRef
  • Google Scholar

• 20

  Cervantes-GomezFLamotheBWoyachJAWierdaWGKeatingMJBalakrishnanKet al. Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner With Ibrutinib in Chronic Lymphocytic Leukemia. Clin Cancer Res (2015) 21(16):3705–15. doi: 10.1158/1078-0432.CCR-14-2809

  • CrossRef
  • Google Scholar

• 21

  HaselagerMVKielbassaKTer BurgJBaxDJFernandesSMBorstJet al. Changes in Bcl-2 Members After Ibrutinib or Venetoclax Uncover Functional Hierarchy in Determining Resistance to Venetoclax in CLL. Blood (2020) 136(25):2918–26. doi: 10.1182/blood.2019004326

  • CrossRef
  • Google Scholar

• 22

  de RooijMFKuilAGeestCRElderingEChangBYBuggyJJet al. The Clinically Active BTK Inhibitor PCI-32765 Targets B-Cell Receptor–and Chemokine-Controlled Adhesion and Migration in Chronic Lymphocytic Leukemia. Blood J Am Soc Hematol (2012) 119(11):2590–4. doi: 10.1182/blood-2011-11-390989

  • CrossRef
  • Google Scholar

• 23

  KaterAPSlingerECretenetGMartensAWBalasubramanianSLeversonJDet al. Combined Ibrutinib and Venetoclax Treatment vs Single Agents in the TCL1 Mouse Model for Chronic Lymphocytic Leukemia. Blood Adv (2021) 5(23):5410–4. doi: 10.1182/bloodadvances.2021004861

  • CrossRef
  • Google Scholar

• 24

  DengJIsikEFernandesSMBrownJRLetaiADavidsMS. Bruton’s Tyrosine Kinase Inhibition Increases BCL-2 Dependence and Enhances Sensitivity to Venetoclax in Chronic Lymphocytic Leukemia. Leukemia (2017) 31(10):2075–84. doi: 10.1038/leu.2017.32

  • CrossRef
  • Google Scholar

• 25

  LuPWangSFranzenCAVenkataramanGMcClureRLiLet al. Ibrutinib and Venetoclax Target Distinct Subpopulations of CLL Cells: Implication for Residual Disease Eradication. Blood Cancer J (2021) 11(2):39. doi: 10.1038/s41408-021-00429-z

  • CrossRef
  • Google Scholar

• 26

  TamCSAndersonMAPottCAgarwalRHandunnettiSHicksRJet al. Ibrutinib Plus Venetoclax for the Treatment of Mantle-Cell Lymphoma. N Engl J Med (2018) 378(13):1211–23. doi: 10.1056/NEJMoa1715519

  • CrossRef
  • Google Scholar

• 27

  JainNKeatingMThompsonPFerrajoliABurgerJABorthakurGet al. Ibrutinib Plus Venetoclax for First-Line Treatment of Chronic Lymphocytic Leukemia: A Nonrandomized Phase 2 Trial. JAMA Oncol (2021) 7(8):1213–9. doi: 10.1001/jamaoncol.2021.1649

  • CrossRef
  • Google Scholar

• 28

  HowardDRHockadayABrownJMGregoryWMToddSMunirTet al. A Platform Trial in Practice: Adding a New Experimental Research Arm to the Ongoing Confirmatory FLAIR Trial in Chronic Lymphocytic Leukaemia. Trials (2021) 22(1):1–13. doi: 10.1186/s13063-020-04971-2

  • CrossRef
  • Google Scholar

• 29

  LevinM-DKaterAMattssonMKerstingSRantiJTranHTTet al. Protocol Description of the HOVON 141/VISION Trial: A Prospective, Multicentre, Randomised Phase II Trial of Ibrutinib Plus Venetoclax in Patients With Creatinine Clearance≥ 30 Ml/Min Who Have Relapsed or Refractory Chronic Lymphocytic Leukaemia (RR-CLL) With or Without TP53 Aberrations. BMJ Open (2020) 10(10):e039168. doi: 10.1136/bmjopen-2020-039168

  • CrossRef
  • Google Scholar

• 30

  JainNKeatingMJThompsonPABurgerJAFerrajoliAEstrovZEet al. Combined Ibrutinib and Venetoclax in Patients With Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL). Washington, DC: American Society of Hematology (2019).

  • Google Scholar

• 31

  TamCSSiddiqiTAllanJNKippsTJFlinnIWKussBJet al. Ibrutinib (Ibr) Plus Venetoclax (Ven) for First-Line Treatment of Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL): Results From the MRD Cohort of the Phase 2 CAPTIVATE Study. Blood (2019) 134(Supplement_1):35. doi: 10.1182/blood-2019-121424

  • CrossRef
  • Google Scholar

• 32

  WierdaWGDorritieKAMunozJStephensDMSolomonSRGillenwaterHHet al. Ibrutinib (Ibr) Plus Venetoclax (Ven) for First-Line Treatment of Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL): 1-Year Disease-Free Survival (DFS) Results From the MRD Cohort of the Phase 2 CAPTIVATE Study Presented at: The 2020 ASH Annual Meeting and Exposition; December 5-8, 2020 Abstract #123. Elsevier (2020).

  • Google Scholar

• 33

  KaterAOwenCMorenoCFollowsGMunirTLevinM-Det al. Fixed-Duration Ibrutinib and Venetoclax (I+V) Versus Chlorambucil Plus Obinutuzumab (Clb+O) for First-Line (1L) Chronic Lymphocytic Leukemia (CLL): Primary Analysis of the Phase 3 GLOW Study Vol. 330172. EHA Library (2021). p. LB1902.

  • Google Scholar

• 34

  BeroukhimRMermelCHPorterDWeiGRaychaudhuriSDonovanJet al. The Landscape of Somatic Copy-Number Alteration Across Human Cancers. Nature (2010) 463(7283):899–905. doi: 10.1038/nature 08822

  • Google Scholar

• 35

  SmitLAHallaertDYSpijkerRde GoeijBJaspersAKaterAPet al. Differential Noxa/Mcl-1 Balance in Peripheral Versus Lymph Node Chronic Lymphocytic Leukemia Cells Correlates With Survival Capacity. Blood (2007) 109(4):1660–8. doi: 10.1182/blood-2006-05-021683

  • CrossRef
  • Google Scholar

• 36

  LongoPGLaurentiLGobessiSSicaSLeoneGEfremovDG. The Akt/Mcl-1 Pathway Plays a Prominent Role in Mediating Antiapoptotic Signals Downstream of the B-Cell Receptor in Chronic Lymphocytic Leukemia B Cells. Blood J Am Soc Hematol (2008) 111(2):846–55. doi: 10.1182/blood-2007-05-089037

  • CrossRef
  • Google Scholar

• 37

  BojarczukKSasiBKGobessiSInnocentiIPozzatoGLaurentiLet al. BCR Signaling Inhibitors Differ in Their Ability to Overcome Mcl-1–Mediated Resistance of CLL B Cells to ABT-199. Blood J Am Soc Hematol (2016) 127(25):3192–201. doi: 10.1182/blood-2015-10-675009

  • CrossRef
  • Google Scholar

• 38

  GuièzeRLiuVMRosebrockDJourdainAAHernández-SánchezMZuritaAMet al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell (2019) 36(4):369–84.e13. doi: 10/1016/j.ccell.2019.08.005

  • Google Scholar

• 39

  KapoorIBodoJHillBTHsiEDAlmasanA. Targeting BCL-2 in B-Cell Malignancies and Overcoming Therapeutic Resistance. Cell Death Dis (2020) 11(11):1–11. doi: 10.1038/s41419-020-03144-y

  • CrossRef
  • Google Scholar

• 40

  JayappaKDGordonVLMorrisCGWilsonBShettyBDCiosKJet al. Extrinsic Interactions in the Microenvironment In Vivo Activate an Antiapoptotic Multidrug-Resistant Phenotype in CLL. Blood Adv (2021) 5(17):3497–510. doi: 10.1182/bloodadvances.2020003944

  • CrossRef
  • Google Scholar

• 41

  JayappaKDPortellCAGordonVLCapaldoBJBekiranovSAxelrodMJet al. Microenvironmental Agonists Generate De Novo Phenotypic Resistance to Combined Ibrutinib Plus Venetoclax in CLL and MCL. Blood Adv (2017) 1(14):933–46. doi: 10.1182/bloodadvances.2016004176

  • CrossRef
  • Google Scholar

• 42

  TronAEBelmonteMAAdamAAquilaBMBoiseLHChiarparinEet al. Discovery of Mcl-1-Specific Inhibitor AZD5991 and Preclinical Activity in Multiple Myeloma and Acute Myeloid Leukemia. Nat Commun (2018) 9(1):5341. doi: 10.1038/s41467-018-07551-w

  • CrossRef
  • Google Scholar

• 43

  CaenepeelSBrownSPBelmontesBMoodyGKeeganKSChuiDet al. AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination With Established Therapies. Cancer Discovery (2018) 8(12):1582–97. doi: 10.1158/2159-8290.CD-18-0387

  • CrossRef
  • Google Scholar

• 44

  YiXSarkarAKismaliGAslanBAyresMIlesLRet al. AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia. Clin Cancer Res (2020) 26(14):3856–67. doi: 10.1158/1078-0432.CCR-19-1397

  • CrossRef
  • Google Scholar

• 45

  KotschyASzlavikZMurrayJDavidsonJMaragnoALLe Toumelin-BraizatGet al. The MCL1 Inhibitor S63845 Is Tolerable and Effective in Diverse Cancer Models. Nature (2016) 538(7626):477–82. doi: 10.1038/nature19830

  • CrossRef
  • Google Scholar

• 46

  LiZHeSLookAT. The MCL1-Specific Inhibitor S63845 Acts Synergistically With Venetoclax/ABT-199 to Induce Apoptosis in T-Cell Acute Lymphoblastic Leukemia Cells. Leukemia (2019) 33(1):262–6. doi: 10.1038/s41375-018-0201-2

  • CrossRef
  • Google Scholar

• 47

  ChenLSKeatingMJWierdaWGGandhiV. Induction of Apoptosis by MCL-1 Inhibitors in Chronic Lymphocytic Leukemia Cells. Leukemia Lymphoma (2019) 60(12):3063–6. doi: 10.1080/10428194.2019.1622098

  • CrossRef
  • Google Scholar

• 48

  Cervantes-GomezFKumar PatelVBosePKeatingMJGandhiV. Decrease in Total Protein Level of Bruton’s Tyrosine Kinase During Ibrutinib Therapy in Chronic Lymphocytic Leukemia Lymphocytes. Leukemia (2016) 30(8):1803–4. doi: 10.1038/leu.2016.129

  • CrossRef
  • Google Scholar

• 49

  WeiMCZongWXChengEHLindstenTPanoutsakopoulouVRossAJet al. Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death. Science (2001) 292(5517):727–30. doi: 10.1126/science.1059108

  • CrossRef
  • Google Scholar

• 50

  PatelVBalakrishnanKBibikovaEAyresMKeatingMJWierdaWGet al. Comparison of Acalabrutinib, A Selective Bruton Tyrosine Kinase Inhibitor, With Ibrutinib in Chronic Lymphocytic Leukemia Cells. Clin Cancer Res (2017) 23(14):3734–43. doi: 10.1158/1078-0432.CCR-16-1446

  • CrossRef
  • Google Scholar

• 51

  PatelVKLamotheBAyresMLGayJCheungJPBalakrishnanKet al. Pharmacodynamics and Proteomic Analysis of Acalabrutinib Therapy: Similarity of on-Target Effects to Ibrutinib and Rationale for Combination Therapy. Leukemia (2018) 32(4):920–30. doi: 10.1038/leu.2017.321

  • CrossRef
  • Google Scholar

• 52

  YangQModiPKorkutAFernandesSMHannaJBrownJRet al. Changes in Bcl-2 Family Protein Profile During Idelalisib Therapy Mimic Those During Duvelisib Therapy in Chronic Lymphocytic Leukemia Lymphocytes. JCO Precis Oncol (2017) 1. doi: 10.1200/PO.17.00025

  • CrossRef
  • Google Scholar

• 53

  PatelVMBalakrishnanKDouglasMTibbittsTXuEYKutokJLet al. Duvelisib Treatment is Associated With Altered Expression of Apoptotic Regulators That Helps in Sensitization of Chronic Lymphocytic Leukemia Cells to Venetoclax (ABT-199). Leukemia (2017) 31(9):1872–81. doi: 10.1038/leu.2016.382

  • CrossRef
  • Google Scholar

• 54

  PonaderSBurgerJA. Bruton’s Tyrosine Kinase: From X-Linked Agammaglobulinemia Toward Targeted Therapy for B-Cell Malignancies. J Clin Oncol (2014) 32(17):1830–9. doi: 10.1200/JCO.2013.53.1046

  • CrossRef
  • Google Scholar

• 55

  ChenLSBosePCruzNDJiangYWuQThompsonPAet al. A Pilot Study of Lower Doses of Ibrutinib in Patients With Chronic Lymphocytic Leukemia. Blood (2018) 132(21):2249–59. doi: 10.1182/blood-2018-06-860593

  • CrossRef
  • Google Scholar

• 56

  KrajewskaMKrajewskiSEpsteinJIShabaikASauvageotJSongKet al. Immunohistochemical Analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 Expression in Prostate Cancers. Am J Pathol (1996) 148(5):1567–76.

  • Google Scholar

• 57

  OpfermanJTLetaiABeardCSorcinelliMDOngCCKorsmeyerSJ. Development and Maintenance of B and T Lymphocytes Requires Antiapoptotic MCL-1. Nature (2003) 426(6967):671–6. doi: 10.1038/nature02067

  • CrossRef
  • Google Scholar

• 58

  NijhawanDFangMTraerEZhongQGaoWDuFet al. Elimination of Mcl-1 Is Required for the Initiation of Apoptosis Following Ultraviolet Irradiation. Genes Dev (2003) 17(12):1475–86. doi: 10.1101/gad.1093903

  • CrossRef
  • Google Scholar

• 59

  WengCLiYXuDShiYTangH. Specific Cleavage of Mcl-1 by Caspase-3 in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis in Jurkat Leukemia T Cells. J Biol Chem (2005) 280(11):10491–500. doi: 10.1074/jbc.M412819200

  • CrossRef
  • Google Scholar

• 60

  HillmenPRawstronACBrockKMuñoz-VicenteSYatesFJBishopRet al. Ibrutinib Plus Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia: The CLARITY Study. J Clin Oncol (2019) 37(30):2722. doi: 10.1200/JCO.19.00894

  • CrossRef
  • Google Scholar

• 61

  ZhouQLiTPriceDH. RNA Polymerase II Elongation Control. Annu Rev Biochem (2012) 81:119–43. doi: 10.1146/annurev-biochem-052610-095910

  • CrossRef
  • Google Scholar

• 62

  TongWGChenRPlunkettWSiegelDSinhaRHarveyRDet al. Phase I and Pharmacologic Study of SNS-032, A Potent and Selective Cdk2, 7, and 9 Inhibitor, in Patients With Advanced Chronic Lymphocytic Leukemia and Multiple Myeloma. J Clin Oncol (2010) 28(18):3015–22. doi: 10.1200/JCO.2009.26.1347

  • CrossRef
  • Google Scholar

• 63

  ByrdJCLinTSDaltonJTWuDPhelpsMAFischerBet al. Flavopiridol Administered Using a Pharmacologically Derived Schedule Is Associated With Marked Clinical Efficacy in Refractory, Genetically High-Risk Chronic Lymphocytic Leukemia. Blood (2007) 109(2):399–404. doi: 10.1182/blood-2006-05-020735

  • CrossRef
  • Google Scholar

• 64

  ChenRKeatingMJGandhiVPlunkettW. Transcription Inhibition by Flavopiridol: Mechanism of Chronic Lymphocytic Leukemia Cell Death. Blood (2005) 106(7):2513–9. doi: 10.1182/blood-2005-04-1678

  • CrossRef
  • Google Scholar

• 65

  ChenRGuoLChenYJiangYWierdaWGPlunkettW. Homoharringtonine Reduced Mcl-1 Expression and Induced Apoptosis in Chronic Lymphocytic Leukemia. Blood (2011) 117(1):156–64. doi: 10.1182/blood-2010-01-262808

  • CrossRef
  • Google Scholar

• 66

  LucasDMEdwardsRBLozanskiGWestDAShinJDVargoMAet al. The Novel Plant-Derived Agent Silvestrol has B-Cell Selective Activity in Chronic Lymphocytic Leukemia and Acute Lymphoblastic Leukemia In Vitro and In Vivo. Blood (2009) 113(19):4656–66. doi: 10.1182/blood-2008-09-175430

  • CrossRef
  • Google Scholar