Inhibition of cyclin‐dependent kinase 9 synergistically enhances venetoclax activity in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non‐Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co‐expression of other anti‐apoptotic proteins such as MCL‐1, which is associated with both intrinsic and acquired resistance to venetoclax in B‐cell malignancies. One strategy for neutralizing MCL‐1 and other short‐lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL‐2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL‐1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib.

Concurrent overexpression of BCL-2 and MCL-1 has been described in clinical DLBCL samples [17] and other subtypes of NHL [18]. The co-expression of anti-apoptotic proteins in NHL samples suggests that therapeutic targeting of one of these proteins exclusively may lack efficacy in the clinic. Venetoclax shows high objective response rates in CLL as a monotherapy [19][20][21]. However, the single-agent efficacy of venetoclax has not been as extensive and durable in patients with relapsed or refractory NHL [22], a collection of B-cell malignancies where MCL-1 is established as a factor driving resistance to venetoclax [17,[23][24][25]. Given the co-expression of both BCL-2 and MCL-1 in subsets of NHL such as MCL [18], we hypothesized that simultaneous targeting of these proteins would afford increased efficacy in this disease.
CDK9 is a serine/threonine kinase that forms the catalytic core of the positive transcription elongation factor b. CDK9 phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase-II (Pol-II), which is required for transcript elongation [26][27][28]. This can be blocked through small molecule CDK9 inhibition, where binding to the CDK9 subunit prevents it from interacting with the C-terminal domain of RNA Pol-II.
CDK9 inhibition has the most immediate effect on proteins with rapid turnover rates such as XIAP and MCL-1 [29]. Consequently, CDK9 inhibition in cell lines that depend upon functional MCL-1 for survival results in the rapid induction of apoptosis [30,31].
In this study, the combined effects of venetoclax with the selective CDK9 inhibitors A-1467729 and A-1592668 [31] were investigated in MCL cell lines and primary samples representing de novo and relapsed cases. We observed potent combination activity in both in vitro and in vivo models. Further studies deciphering the mechanism of the combined effects supported the hypothesis that MCL-1 downregulation via CDK9 inhibition can sensitize MCL cells to BCL-2 inhibition.

Materials and treatment
Jeko-1 cells were from DSMZ. Mino and JVM-2 cells were from ATCC.
CCMCL1 cells were obtained as described previously [32]. Lymphoma biopsies were obtained following protocols approved by the Institu-

Immunohistochemistry
Briefly, 4 μm of formalin-fixed paraffin-embedded tissue sections were used for IHC staining. Resources of antibodies, equipment, and detection systems are summarized in Table S2.
The diameters of subcutaneous tumors were measured and tumor volume (mm 3 ) was calculated as described previously [32] at the endpoints of the study or when tumors reached the maximum size allowed by IACUC.

Statistical analysis
The interaction between drugs was examined according to the Chou and Talalay method [36,37]

Responses of MCL cell lines to venetoclax and CDK9 inhibitors
NHL is characterized by heterogeneous expression of the BCL-2 family of apoptosis regulators [18,17], which may in part reflect the limited activity of venetoclax in DLBCL patients [22]. To understand whether this expression profile is also reflective of MCL, we first

CDK9 inhibitor synergistically enhances venetoclax activity and causes rapid loss of MCL-1
Despite the different responses to each single agent, venetoclax plus either A-1467729 or A-1592668 resulted in synergistic apoptosis in all MCL cell lines tested ( Figure 2A; Figure S1A,B) including JVM-2 cells, which were less sensitive to each single-agent treatment. In concordance with the single-agent data in which JVM-2 cells were the least sensitive cells, the overall apoptotic effect of the combination was also lower in this cell line. The combination of venetoclax with A-1467729 strongly enhanced PARP cleavage in all cell lines and was associated with the mechanism-based downregulation of p-RNApol-II (Ser2) and MCL-1 whereas BCL-2 levels were unaffected ( Figure 2B).

Direct MCL-1 inhibition has similar effects as CDK9 inhibition
To confirm that CDK9 inhibitor-induced reduction of MCL-1 protein contributed to the synergy between venetoclax and A-1467729/

CDK9 inhibitor or MCL-1 inhibitor enhances venetoclax activity in primary cells
Seven primary MCL cases (Table 1)

Immunohistochemistry
To examine the correlation between BCL-2 and MCL-1 expression and sensitivity of primary samples to the treatment, we performed immunohistochemistry staining on cases with available tissues (1, 2, and 6) or cytospin (case 3) blocks. As shown in Figure 4, all cases were positive for cyclin D1. Concurrent expression of BCL-2 and MCL-1 were detected in all cases but with diverse expression pattern among the cases. Compared to BCL-2 or MCL-1 expression levels, BCL-X L protein was either undetectable (cases 3 and 6) or relatively weak expression. BIM was strongly positive in cases 1 and 2, weakly positive in case 6, and undetectable in case 3. This lack of BIM expression in case 3 was consistent with our previous SNP microarray data [32], which demonstrated BIM deletion on chromosome 2q13. As summarized in Table 1, case 3 was a relapsed MCL with multiple drug failures and a complex karyotype, including MYC rearrangement. Together, this may explain the lack of response to venetoclax. Compared to cases 1 and 2, case 6 displayed weak BCL-2 but stronger MCL-1 expression, which is consistent with the weak response of these cells to venetoclax yet robust sensitivity to the combination of venetoclax plus MCL-1 inhibitor A-1210477 ( Figure 3A).

Antitumor efficacy of venetoclax plus CDK9 inhibition in a xenograft model of MCL
To examine the effects of concomitant inhibition of BCL-2 and CDK9 in vivo, we evaluated the combination of venetoclax plus A-1592668 or dinaciclib, a pan CDK inhibitor that has been tested in clinical trials for advanced malignancies [38,39]. NSG mice were inoculated with Jeko-1 cells, then randomly divided into six groups with six mice per group when tumors reached 25 mm 3 . Venetoclax was dosed at 50 mg/kg/day, while A-1592668 and dinaciclib were dosed at 4 mg/kg or 30 mg/kg twice a week, respectively. All agents were well tolerated as monotherapy or in combination. As shown in Figure 5, treatment with each single agent reduced the tumor burden compared to the control. Venetoclax plus A-1592668 or venetoclax plus dinaciclib co-treatment robustly inhibited the growth of Jeko-1 tumors during 4 weeks of treatment and provided a significant survival advantage over either agent alone or the vehicle control arm (P < .05). These data demonstrate that the combination of venetoclax plus CDK9 inhibition is effective against MCL in vivo.

DISCUSSION
Venetoclax is a potent and selective BCL-2 inhibitor that has demonstrated high objective response rates as a monotherapy in patients with CLL, a B-cell malignancy that possesses a strong dependence on BCL-2 for survival. While venetoclax has shown objective responses in a number of NHL subtypes as monotherapy, the overall response rates and durability were not as high as in CLL [22,19]. Co-expression and codependence on multiple BCL-2 family proteins in NHLs may act as a and subsequently driving apoptosis in tumor cells that depend upon these factors for survival [17,31,25]. is combined with agents that inhibit MCL-1 or repress its expression.

F I G U R E 3 Interactions of venetoclax plus CDK9 inhibitors or venetoclax plus MCL-1 inhibitor in primary MCL cells. A, Primary MCL cells were co-treated for 5 h with venetoclax plus
Furthermore, the in vitro efficacy derived from this combination was recapitulated in a mouse xenograft model. These findings support the clinical evaluation of venetoclax in combination with CDK9 or MCL-1 inhibitors in MCL, including ibrutinib-resistant MCL.

ACKNOWLEDGMENTS
The authors would like to thank Daniel Lindner and Yvonne Parker for their technical support in handling NSG mice. The authors also thank Neetha Parameswaran for her critical reading of this manuscript.
Financial support for this research was provided by AbbVie Inc.

AUTHOR CONTRIBUTIONS
XZ and EDH are the principal investigators and take primary responsibility for the manuscript. XZ, RC, and LD performed laboratory work for this study. JB performed the analysis of data. XZ and EDH coordinated the research. XZ, JB, AS, DP, and EDH contributed to the experimental design and writing of the manuscript.