New Strategies Aim at Survival Protein MCL-1

Myeloid cell leukemia 1 (MCL-1), a key signaling protein in cancer cell survival pathways, has emerged as an intriguing target for anticancer drug development as researchers turn their attention to strategies directed at evasion of apoptosis, a distinguishing characteristic of cancer.

Figure. Signaling Networks Active in Apoptosis

As part of the B-cell lymphoma-2 (BCL-2) family of proteins, MCL-1 is among the central coordinators of apoptosis. The ability to evade apoptosis, whereby cancer cells thrive amid the stresses of oncogenesis, is characterized as a hallmark of cancer because it is one of the unique acquired abilities that allow the malignant transformation of a normal cell.In 2016, venetoclax (Venclexta) became the first BCL-2 inhibitor to gain FDA approval, with an indication for patients with relapsed and refractory chronic lymphocytic leukemia (CLL). Despite this success, resistance to BCL-2 inhibition develops and is frequently mediated by alterations in other BCL-2 protein family members. In particular, MCL-1 overexpression is one of the most common molecular aberrations observed in many types of cancer.

The development of MCL-1 inhibitors has proved challenging but at last appears to be bearing fruit. Both direct inhibition through small-molecule MCL-1—specific inhibitors and indirect targeting through blockade of cyclindependent kinase 9 (CDK9) are showing promise in the early stages of development in a range of cancer types. Additionally, pairing MCL-1 inhibitors with venetoclax displays synergistic activity and could help mitigate acquired resistance. They also may be highly effective combined with other targeted drugs and with standard-of-care agents.

Tight Regulation of Cell Death

The next major challenge will be the identification of accurate predictive biomarkers to help guide the optimal clinical application of MCL-1 inhibitors and other drugs targeting this family of survival proteins.Apoptosis is a tightly controlled form of cell death that clears unwanted or damaged cells and maintains tissue homeostasis, without breaching the plasma membrane or releasing potentially damaging cellular contents.

Two major pathways induce apoptosis— extrinsic and intrinsic—which, as their names suggest, are triggered by extra- and intracellular cues, respectively. The intrinsic pathway is conducted primarily by the BCL-2 family, a group of more than a dozen proteins that share conserved sequences known as BCL-2 homology (BH) domains. They can be divided into 3 groups of proteins: antiapoptotic, proapoptotic (BH3-only), and proapoptotic (effector). These proteins interact with one another to create a delicate balance of pro- and antiapoptotic signals that govern the cell’s fate.

The antiapoptotic BCL-2 proteins include BCL-2, after which the family is named, as well as BCL-XL, BCL-W, MCL-1, and BFL/A1. The proapoptotic proteins can be further subdivided into those that share just a BH3 domain: the BH3-only proteins, which include BID, BAD, BIK, PUMA, and NOXA, and the effector proteins— BAK, BAX, and BOK.

In healthy cells, the proapoptotic effector proteins are bound and sequestered by the antiapoptotic proteins, keeping their activity in check. In response to stimuli such as DNA lesions, mitotic defects, and oxidative stress, the BH3-only proapoptotic proteins are upregulated. They bind to the antiapoptotic proteins and block their suppression of the proapoptotic effector proteins, particularly BAK and BAX. Several of the BH3-only proteins can also directly activate BAK and BAX.

BAK and BAX subsequently oligomerise and form pores within the mitochondrial membrane, allowing cytochrome c to be released into the cytoplasm. Cytochrome c drives the formation of the apoptosome, a protein complex that activates caspase-9, which in turn activates other effector caspases.

A Cancer Hallmark

Caspases are proteases that break down key intracellular proteins, driving characteristic apoptotic outcomes, including fragmentation of the DNA, shrinking of the cell, and blebbing of the membrane. It culminates in the cell breaking up into apoptotic bodies that are engulfed by the phagocytes of the immune system^1-5 (Figure⁶).The apoptotic program is a vital component in the development and maintenance of a healthy organism. However, disruption of the program’s checks and balances can have pathologic effects. An acquired ability to evade apoptosis is a characteristic of cancer cells, permitting them to grow unchecked, even in the presence of cancerinduced DNA damage or cellular stress, and can help them resist anticancer therapy.

One of the central coordinators of apoptosis is the tumor suppressor protein p53. Among its myriad functions is the detection of the cellular conditions required to trigger apoptosis, such as DNA damage, earning it the nickname “guardian of the genome.” A mutated or missing TP53 gene is one of the most common ways by which tumor cells evade apoptosis.

Many members of the BCL-2 protein family are also dysregulated in cancer. The family’s eponymous founding member was initially identified as the protein product of a gene that is translocated and overexpressed in a large number of follicular lymphoma cases. High BCL-2 expression levels subsequently have been identified in a wide range of solid tumor types, including small cell lung, breast cancer, prostate, colorectal, and bladder cancers and melanoma, as well as in hematologic malignancies such as acute myeloid leukemia (AML) and multiple myeloma.

Although the ability to evade apoptosis is undoubtedly a hallmark of cancer cells, this does not mean that they have completely switched off this form of cell death. Cancer cells still undergo apoptosis, and blocking apoptotic signaling by itself is not generally sufficient to drive cancer formation.

Targeting BCL-2 With Venetoclax

A developing cancer cell gets subjected to many stresses that should initiate apoptotic signaling; thus, cancer cells develop the ability to suppress apoptosis in response to those specific triggers. This explains why most cancer cells are initially sensitive to chemotherapy and other conventional therapies but eventually become resistant.^3,7-9As central regulators of cancer cell survival, the antiapoptotic members of the BCL-2 protein family have drawn research interest for anticancer therapy. Efforts to target BCL-2 began in the 1990s, and a range of approaches have been tested.

The greatest success has been achieved with small-molecule inhibitors that function as BH3 mimetics. These drugs are designed to mimic the BH3 domain of BH3-only proapoptotic BCL-2 family members and their function in sequestering the antiapoptotic proteins. First to reach clinical trials were the broad-spectrum drugs ABT-737 and navitoclax (ABT-263), which target multiple BH3-only proteins. Their use was hindered by the development of thrombocytopenia that is thought to result from the inhibition of BCL-XL, which plays a role in platelet survival.

Significant success ultimately came in the form of a specific BCL-2 inhibitor, venetoclax, which did not elicit thrombocytopenia and demonstrated substantial activity in patients with CLL. Venetoclax received accelerated approval for this indication based on the results of a single-arm clinical trial in 107 patients with CLL who had chromosome 17p deletion and had received at least 1 prior therapy, which demonstrated overall response rates (ORRs) in excess of 70%.¹⁰

In July 2017, venetoclax received a breakthrough therapy designation for use in combination with low-dose cytarabine in treatment-naïve elderly patients with AML who are ineligible for intensive chemotherapy. This was based on the findings of the phase I/II M14-387 study. In the initial dose-escalation phase, 18 patients received oral venetoclax at a dose of 600 mg to 800 mg once daily on days 1 to 28 in combination with low-dose cytarabine at 20 mg/m² daily on days 1 to 10 of each 28-day cycle. The recommended phase II dose of 600 mg was then administered in the expansion phase to another 53 patients, in addition to 8 patients from the initial cohort. The ORR was 61%, including a complete remission (CR) rate of 21%, a CR with incomplete hematologic recovery rate of 33%, and a partial remission rate of 7%.¹¹

Venetoclax also has a breakthrough therapy designation for patients with treatment-naïve AML who are ineligible to receive standard induction chemotherapy, and it has demonstrated efficacy in patients with heavily treated multiple myeloma.¹²

MCL-1 Presents a Tough Target

Despite its success, venetoclax, like any targeted cancer therapy, encounters resistance. These mechanisms are being studied in an effort to circumvent resistance as it emerges. One of the central ways that resistance develops involves the compensatory upregulation of other antiapoptotic BCL-2 family proteins—in particular, MCL-1.^13,14MCL-1 has emerged as a promising drug target for reasons beyond its role in venetoclax resistance. Overexpression of MCL-1 proteins is a common feature of many tumor types. Aberrations in the MCL-1 gene that encodes the proteins are among the most common genetic defects in lung, breast, prostate, pancreatic, ovarian, and cervical cancers, as well as in melanoma and some hematologic malignancies. Indeed, MCL-1 is among the top 10 types, and MCL-1 aberrations have been shown to play a role in resistance to many anticancer therapies beyond venetoclax.^2,15-17

New Agents in Development

However, the development of drugs that can target MCL-1 activity has proved particularly challenging. Several BCL-2 inhibitors, which included MCL-1 among their multiple targets, did not advance after being evaluated in clinical trials. These included obatoclax (GX15-070) and AT-101 (gossypol), which were well tolerated in clinical studies but demonstrated limited efficacy. The development of obatoclax was discontinued in 2012, although several clinical trials of AT-101 continue.¹⁸ Additionally, a variety of BH3 mimetics that appeared to be potent and specific inhibitors of MCL-1 turned out not to be sufficiently potent or selective.¹⁹More recently, several novel drugs have emerged that more closely fit the criteria for selective MCL-1 inhibitors and have reached the early stages of clinical testing, with several others in preclinical development (TABLE). Early and preclinical data have been very promising, including findings presented at this year’s American Association for Cancer Research (AACR) Annual Meeting 2018.

In preclinical models of multiple myeloma and AML, AstraZeneca’s AZD5991 demonstrated potent antitumor activity, including complete tumor regression in several cases. It also demonstrated synergistic activity with standard-of-care therapies.²⁰ A phase I clinical trial is under way in patients with hematologic malignancies.

Making an Indirect Hit Through CDK

Although most ongoing clinical trials focus on evaluating MCL-1 inhibitors in hematologic malignancies, particularly those that are commonly asserted to be MCL-1—dependent, preclinical data suggest that this strategy may also have efficacy in patients with solid tumors.^21,22 Researchers at the Walter and Eliza Hall Institute in Melbourne, Australia, have been studying the compound S63845, jointly developed by the pharmaceutical companies Servier and Vernalis. It has demonstrated antitumor efficacy in diverse tumor models, including melanoma and cancers of the lung and breast.²³Another mechanism gaining attention indirectly targets MCL-1 through CDKs, a group of serine-threonine kinases that are dependent upon the binding of a cyclin protein for activation. Due to their important role in regulating the cell cycle, CDKs have proved a promising anticancer drug target in their own right in recent years, and several inhibitors of the CDK4 and 6 proteins are approved by the FDA.

CDK9 plays a vital role in the transcription of DNA into RNA—specifically, the elongation stage—through its phosphorylation and regulation of the key enzyme RNA polymerase II. MCL-1 is a downstream transcriptional target of CDK9; several studies have shown that CDK inhibition downregulates MCL-1 levels and that the resulting antiapoptotic effect may be an important component of the mechanism of action of CDK inhibitors that target CDK9.^24,25

A number of novel drugs in this category are being explored as single agents and in combination therapies (Table). Results of a first-in-human study of CYC065 were presented at AACR 2018. In a phase I trial in patients with advanced cancers, CYC065 was administered as a 4-hour infusion every 3 weeks at 7 different dose levels to 26 patients with various forms of heavily pretreated cancers.26 The maximum tolerated dose was 192 mg/m², which was selected as the recommended phase II dose. Among 13 patients treated at this dose, stable disease was the best response observed in 46%, including those with larynx neuroendocrine carcinoma, ovarian adenocarcinoma, and uterine carcinosarcoma; the longest response lasted around 1 year.

Combinations are Explored

Dose-limiting toxicities included reversible neutropenia, thrombocytopenia, febrile neutropenia, diarrhea, hypomagnesemia, white blood cell lysis syndrome, and liver enzyme elevations. The most common adverse events included constipation, diarrhea, decreased appetite, dehydration, fatigue, and nausea and vomiting, with most being mild to moderate in intensity.Although single-agent activity has been observed, MCL-1 inhibitors, both direct and indirect, are likely to prove most effective as part of combination regimens. Identifying the most rational combinations and optimizing their use is the next major challenge for MCL-1—targeted drug development.

Table. Selected Clinical Trials of Agents Directed at MCL-1 Activity

Several preclinical studies demonstrate synergistic activity for MCL-1 inhibition in combination regimens pairing venetoclax with CDK9 inhibitors, including alvocidib, voruciclib, and dinaciclib. The results of preclinical studies of 2 novel CDK9 inhibitors, AZD4573 and CYC065, were presented at AACR 2018. Both demonstrated synergistic activity with venetoclax in hematologic cancer models or cell lines, and AZD4573 also showed synergy with other targeted agents.^27,28Additionally, preclinical findings indicate that an MCL-1 inhibitor, AMG 176, reduced the tumor burden by 69% in mouse models of AML as a single agent, but synergistic activity drove complete inhibition of tumor growth when it was combined with venetoclax.²⁹

Meanwhile, promising results have been observed with CDK9 inhibitors and other targeted therapies or chemotherapy. Alvocidib, which is being developed by Tolero Pharmaceuticals, demonstrated markedly enhanced antitumor activity when combined with standard cytarabine and daunorubicin (7 + 3 regimen) in AML xenograft models, according to results presented at AACR 2018.30 The 3-drug combination more than doubled the induction of apoptosis compared with any of the drugs as single agents, suppressed MCL-1 expression, and greatly inhibited tumor growth.

The Search for Biomarkers

Alvocidib is being evaluated in a phase II trial in patients with relapsed or refractory MCL-1— dependent AML in combination with cytarabine and mitoxantrone (NCT02520011) and in a phase I study in combination with 7 + 3 in newly diagnosed AML (NCT03298984).Responses to MCL-1 inhibitors vary among studies and across cancer types, which highlights another important challenge: Identifying accurate biomarkers that predict response will be essential to enhance their clinical activity.^8,31

Gene and protein expression levels of the BCL-2 family members have been proposed as potential biomarkers but proved inconsistent. In a study of S63845 in small cell lung cancer cell lines, MCL-1 expression levels predicted response poorly, but the levels of BCL-XL strongly negatively correlated with response.³² Other studies have suggested that the ratio of MCL-1 to BCL-XL expression could be a useful biomarker of response to indirect MCL-1 inhibitors, such as the CDK inhibitor dinaciclib.^24,33

BH3 profiling can identify the precise apoptotic defects in cancer cells and could potentially predict sensitivity to BCL-2 family inhibitors, although this has not yet been clinically validated. This approach takes advantage of the fact that the interactions between the BH3-only proteins and antiapoptotic proteins are highly selective. The mitochondria are isolated from cancer cells and exposed to a series of BH3 domain peptides, and the ability of these peptides to induce mitochondrial membrane permeabilization is then measured.³⁴

Several preclinical studies also have demonstrated MCL-1’s potential use as a prognostic marker, with MCL-1 overexpression, which can be measured by immunohistochemistry or polymerase chain reaction testing, correlating with poor prognosis in a variety of malignancies. This was most recently demonstrated in a study of breast cancer.²⁰ In renal cell carcinoma cells, MCL-1 expression was found to be significantly higher in well-differentiated adenocarcinomas compared with medium or lowly differentiated specimens.³⁵

References

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