Combining proteasome with cell cycle inhibitors: a dual attack potentially applicable to multiple hematopoietic malignancies

Standard chemotherapy regimens are curative in only a subset of hematological malignancies, such as childhood acute lymphoblastic leukemia and certain subgroups of Hodgkin’s and non-Hodgkin’s lymphoma. Moreover, the molecular targets of many cytotoxic chemotherapeutic agents, despite intensive investigation, remain uncertain. Because of a narrow therapeutic index for cytotoxic agents, most regimens combine different classes of agents with non-overlapping toxicities. Nevertheless, significant toxicities are observed.

Against this background, mechanistically-based therapeutic strategies targeting critical molecular pathways implicated in transformation have attracted considerable interest over the course of the last decade. Targeted therapy involves the use of an agent with a clearly defined mechanism that acts specifically on a validated target or biological pathway that, when inactivated, causes transformed cell death, differentiation or loss of proliferation. The prototypical example of targeted therapy was the introduction of imatinib mesylate (Gleevec^®), an inhibitor of the oncogenic mutant Bcr/Abl kinase, which revolutionized the treatment of chronic myeloid leukemia (CML) [1]. The obvious advantage of such approaches is that they may be less toxic than standard cytotoxic therapy, with a higher therapeutic index and potentially greater efficacy. Furthermore, such novel approaches may offer the potential for lowering the threshold for induction of programmed cell death triggered by more standard cytotoxic agents. Unfortunately, CML represents a relatively rare instance in which a transformed cell is addicted to the dysregulated pathway (i.e., Bcr/Abl). In most other malignancies, including hematopoietic malignancies, addiction to a single pathway does not exist. Instead, pre-existing or stimulated compensatory signaling pathways relieve the cell of its addiction, and permit it to survive despite interruption of the original oncogenic stimulus [2]. In addition, the successful application of targeted strategies requires identification of the oncogenic lesion, and developing agents capable of disrupting its function. As a consequence, considerable attention has recently focused on strategies involving the coordinate disruption of complementary signaling and cell cycle pathways, that is, by regimens combining multiple targeted agents [3]. The central hypothesis underlying this concept is that transformed cells may be particularly ill-equipped to escape the lethal effects of dual interruption of survival signaling and/or cell cycle regulatory pathways.

In this context, findings from preclinical studies suggest that disruption of cell cycle events by cyclin-dependent kinase (CDK) inhibitors such as alvocidib may, when combined with inhibition of survival-related pathways, provide an extremely potent stimulus for apoptosis in malignant hematopoietic cells. Alvocidib (NSC 649890, flavopiridol; L86–8275) is a rohitukine alkaloid that inhibits CDKs 1, 2, 4/6, 7 and 9 [4] and, in some cell types, downregulates expression of cyclin D1 [5]. Alvocidib was the first CDK inhibitor to enter the clinical arena. Alvocidib also acts, at least in preclinical studies, as a transcriptional repressor through its capacity to inhibit the CDK9/cyclin T pTEFb complex [6]. In addition, alvocidib induces downregulation of various anti-apoptotic proteins including p21CIP1 [7], cyclin D1, Mcl-1, XIAP and Bag-1. Notably, alvocidib-mediated downregulation of Mcl-1 has been postulated to play a particularly important functional role in the activity of this agent in multiple myeloma [8]. In addition to its ability to induce cell cycle arrest, alvocidib potently triggers apoptosis in tumor cells, particularly those of hematopoietic origin. Interestingly, alvocidib has been shown to act, through inhibition of IκB kinase, as an effective antagonist of the NF-κB pathway [9], which plays an important role in survival of several malignant hematopoietic cell types, including multiple myeloma and acute myeloid leukemia.

Bortezomib (PS-341; Velcade™) is a boronic anhydride proteasome inhibitor, a class of agents that interferes with the function of the 26S catalytic component of the proteasome, which is responsible for the degradation of a wide variety of cellular proteins, including those related to signal transduction, cell cycle regulation and survival [10]. In preclinical studies, proteasome inhibitors have been shown to induce cell death in diverse tumor cell types [11], an effect that may stem from accumulation of pro-apoptotic proteins (e.g., Bax, Bad and Bid), upregulation of p53, or activation of stress-related kinases (e.g., JNK/stress-activated protein kinase). In addition, there is accumulating evidence that the lethal actions of proteasome inhibitors may be related to inactivation of the cytoprotective NF-κB pathway, that is, by blocking the degradation of the NF-κB-inhibitory protein IκBα. Based on preclinical findings, as well as initial results of clinical trials involving bortezomib and alvocidib administered as single agents, a strong rationale exists for combining these agents in patients with multiple myeloma, mantle cell lymphoma, follicular cell lymphoma, and potentially CML. Bortezomib is highly active in patients with multiple myeloma, including disease that is refractory to standard therapy. Alvocidib has been targeted against multiple myeloma in view of its preclinical activity against this disease as well as its ability to downregulate Mcl-1, an important survival factor in myeloma. In mantle cell lymphoma, alvocidib-induced preclinical activity has been associated with downregulation of cyclin D1, which is characteristically dysregulated in this disease. Importantly, preclinical studies demonstrated that bortezomib and alvocidib interact in a highly synergistic manner to induce apoptosis in human leukemia cells in association with downregulation of XIAP and Mcl-1, activation of the stress-related kinase JNK, and inactivation of NF-κB [12]. Furthermore, this regimen has been reported to induce cell death through a Bcl-2-independent mechanism. Similar interactions have been noted in myeloma and lymphoma cell lines. In CML, this drug combination induces apoptosis in imatinib-resistant CML cell lines through both Bcr/Abl dependent and independent mechanisms [13]. Collectively, these findings raise the possibility that a strategy combining a CDK inhibitor like alvocidib with a proteasome inhibitor such as bortezomib may be effective against diverse hematologic malignancies.

From a clinical standpoint, bortezomib has been approved for use in patients with multiple myeloma [14] and in patients with relapsed/refractory mantle cell lymphoma [15]. Alvocidib has been best studied as a single agent in chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia. Results of early clinical trials in which alvocidib was administered by 1- or 24-h infusions were disappointing [16,17]. In vitro studies revealed that protein binding of alvocidib is much higher in the presence of human versus bovine serum. Accordingly, the necessary therapeutic concentration of alvocidib had been underestimated, leading to the development of a revised schedule in which 50% of the alvocidib dose was administered as a 30-min infusion, followed by 50% dose as a 4-h infusion. Results of a Phase I trial in relapsed, symptomatic CLL/small lymphocytic leukemia yielded a 40% response rate, with median progression-free survival of 12 months. The most frequent adverse events were tumor lysis, neutropenia, diarrhea and cytokine-release syndrome. In a Phase II trial, following adjustment of the treatment regimen due to the high incidence of tumor lysis in the Phase I trial, 47% of patients achieved a partial response and 1.6% achieved a complete remission. The tolerability of the regimen was improved and only 2% of patients exhibited evidence of tumor lysis syndrome [18]. To date, single-agent activity in multiple myeloma, mantle cell lymphoma and acute leukemias has been limited [19–21].

Based on the preceding considerations that the established activity (bortezomib) and potential (alvocidib) activity in B-cell malignancies, preclinical evidence of synergism, and the possibility that transformed cells might be particularly susceptible to disruption of multiple signaling pathways, a combination Phase I clinical trial of alvocidib and bortezomib has recently been completed in patients with relapsed or refractory B-cell neoplasms [22]. A total of 16 patients were treated. The maximum tolerated dose was established as 1.3 mg/m² for bortezomib and 30 mg/m² for alvocidib (for both a 30-min bolus and 4-h infusion). Common hematologic toxicities included leukopenia, lymphopenia, neutropenia and thrombocytopenia. No evidence of tumor lysis or cytokine release syndrome was encountered. Common non-hematologic toxicities included fatigue and febrile neutropenia. Significantly, the overall response rate was 44% with two complete responses (12%) and five partial responses (31%), including several patients who had previously been treated with bortezomib.

While the results of this study are encouraging, several questions arose. For example, the issue of whether a hybrid infusional schedule of alvocidib in conjunction with bortezomib offers advantages over a more standard bolus administration schedule remains to be answered. Results from a nearly completed clinical trial in patients with relapsed or refractory B-cell neoplasms involving a bolus schedule of alvocidib administered with bortezomib may help to address this issue. Furthermore, plans for a limited Phase II efficacy trial for this regimen are in development. Another question involves the mechanism by which these agents might interact in vivo. Based upon the preclinical data, it is tempting to speculate that cooperation between alvocidib and bortezomib in interrupting NF-κB signaling and/or triggering stress-related JNK activation may be involved. It is hoped that correlative laboratory studies will answer these questions. Finally, it will be of interest to determine whether dual interruption of survival signaling and cell cycle regulatory pathways by agents such as alvocidib and bortezomib may have broader applicability to other hematologic malignancies for example, CML, for which preclinical evidence of activity exists, and potentially CLL, where alvocidib may be active. With an increased focus on the strategy of coordinately interrupting multiple pathways with targeted agents, answers to these questions may be forthcoming in the years to come.

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