Exploring MCL-1:
A driver of AML blast survival

Acute myeloid leukemia (AML) is a difficult to treat malignancy with heterogeneous molecular and clinical features.1,2 Historically, AML survival rates have lagged behind other hematologic cancers, with a five-year relative overall survival rate of 27.4% compared with 68.0% in chronic myeloid leukemia or 71.0% in acute lymphocytic leukemia.3

2007 to 2013 five-year relative survival rates3 Click to expand

The elusiveness of durable remission after treatment may stem from the multifaceted and complex nature of blast cell populations within various subtypes of AML.2,4 AML can be characterized based on several factors, including genetically determined prognostic risk, surface markers, or biological aberrations such as signaling pathway dysregulation.5-7

As a result, a new generation of targeted therapies in AML has emerged, some of which are based on risk features.5

See beyond mutation status

Identifying the molecular characteristics of AML blast populations is crucial for target-based therapy.

Emerging research has identified several clinically actionable biological aberrations, some of which are drivers of disease progression.2

Click to learn about the heterogeneous progression of AML.

The complexity of AML

Genetic ProfileAML is a heterogeneous condition with varying genetic expression within blast cell populations.6 Identifying patterns of molecular mutations can provide prognostic information and may help inform treatment decisions.

Mutation status such as FLT3, NPM1, and IDH1/2 distinguish AML blasts
Mutation status may vary between initial diagnosis and relapse.5 Click to expand

Surface Markers5,8AML cells can also be characterized by protein expression, including cell surface markers such as CD33 or CD123. Ligand or receptor proteins that are preferentially expressed in leukemic blasts may be suitably targeted using antibody-based approaches.5

Pathway
Dysregulation5,9,10
Another characterization of AML is based on the dysregulation of signaling pathways, which may occur downstream of genetic mutations or protein overexpression. Aberrant signaling may lead to disruption in cellular processes that are normally tightly regulated, thus driving disease progression.5

Targeting Apoptotic Pathways
The BCL-2 family of proteins

Apoptosis, or programmed cell death, is a vital cellular process that contributes to tissue homeostasis by balancing controlled proliferation.11 It is predominantly regulated by the BCL-2 family, a group of proteins that either promote or suppress the apoptotic signaling process.11,12

BCL-2 family of proteins

The BCL-2 family proteins vary in their expression and activity but can generally be categorized into three groups.

1
Multidomain
pro-apoptotic proteins
(pore forming)12:
Proteins such as BAK, BAX, and BOK form pores in the mitochondrial membrane, releasing cytochrome c and other mitochondrial intermembrane components, leading to the activation of caspases and initiating apoptosis.12,13
2
BH3-only
pro-apoptotic proteins:
BH3-only proteins are induced by cellular stress and apoptotic signals through transcriptional regulation and/or post translational modification. Once activated, these molecules promote the activation and oligomerization of pro-apoptotic effector proteins, BAX and BAK.11
They can be subdivided into 2 groups:
Sensitizers5 NOXA, BMF, BAD, and other BH3-only sensitizers bind to and neutralize anti-apoptotic family members, liberating pro-apoptotic proteins BAK, BAX, and BOK to mediate mitochondrial permeabilization, leading to apoptosis5,12
Activators5 BIM, BID, PUMA, and other BH3-only activators bind to both pro-apoptotic and anti-apoptotic proteins, enabling pro-apoptotic proteins and neutralizing anti-apoptotic proteins5,12
3
Multidomain
anti-apoptotic
proteins5,7,13:
This group binds to and sequesters multidomain pro-apoptotic and BH3-only proteins, blocking apoptosis13

The apoptotic cascade

Normally, all three groups of apoptotic proteins balance and regulate one another, ensuring the survival of healthy cells and the death of damaged cells.13,15

The apoptotic cascade begins when DNA damage triggers a series of upstream responses, which are detected by the BH3-only group. BH3-only proteins are then upregulated, anti-apoptotic proteins are downregulated, and the balance between anti- and pro-apoptotic BCL-2 proteins shifts. The imbalance prompts the activation of pro-apoptotic proteins like BAK, BAX, or BOK.16

Once this occurs, one of two processes take place:

BAK, BAX, BOK, and other pro-apoptotic proteins directly permeabilize the mitochondrial outer membrane, releasing cytochrome c and other mitochondrial intermembrane components, leading to the activation of caspase-9, initiating apoptosis16
or
Anti-apoptotic proteins such as BCL-2, BCL-XL, and MCL-1 bind to and sequester activated BH3-only proteins as well as pro-apoptotic proteins such as BAX and BAK, neutralizing the apoptotic cascade and inhibiting cell death13,14
or

If the expression of anti-apoptotic proteins becomes dysregulated, aberrant proliferation, dedifferentiation, and other abnormal cellular processes may occur. This mechanism may be found in the pathogenesis of many hematologic malignancies, including AML16

How MCL-1 drives
AML blast survival

Myeloid cell leukemia 1, or MCL-1, is an anti-apoptotic member of the BCL-2 family.11 Though similar in structure to other anti-apoptotic BCL-2 proteins, MCL-1 has specific cellular functions and affinities.13,14

MCL-1 is essential for early embryonic development and survival of multiple cell lineages including lymphocytes, hematopoietic stem cells, neutrophils and neurons.7,11 However, overexpression of MCL-1 may result in excessive inhibition of pro-apoptotic proteins and their sensitizers. This provides an avenue for persistent AML blast survival.11,16

MCL-1 constrains apoptosis in two ways:

Directly binding and sequestering pro-apoptotic proteins like BAK, or BAX, inhibiting their ability to permeabilize the mitochondrial outer membrane13

&

Binding to sensitizer proteins like NOXA, PUMA, or BAD, inhibiting their ability to activate BAK, BAX, or BOK13

MCL-1 also regulates mitochondrial metabolism

&

Mitochondrial matrix-localized MCL-1 promotes normal mitochondrial cristae structure, as well as mitochondrial fusion. MCL-1 may also support oxidative phosphorylation, ATP production, and maintenance of mitochondrial membrane potential. These functions may also be important for promoting cancer cell survival, as showcased by the inability of cancer cells to endure the genetic deletion of MCL-1 despite the presence of other anti-apoptotic proteins. It is possible, therefore, that both the anti-apoptotic and mitochondrial functions of MCL-1 synergize to promote leukemic progression.11

Defining MCL-1 dependent AML

Certain subsets of AML rely on MCL-1 for survival14,18

MCL-1 promotion of cancer cell survival can be observed in cancer cells where a subversion of cellular checkpoints has allowed for the increased expression of anti-apoptotic proteins. These proteins "soak up" the increased presence of pro-apoptotic proteins.11 For a subset* of AML, this may be the dominant mechanism of blast survival. In other words, this subset of AML blasts is addicted to MCL-1 and may be susceptible to MCL-1–targeted therapeutic intervention.14,18

*The prevalence of MCL-1–dependent AML
  is under investigation.
Click to see what defines
MCL-1–dependent AML.

Enabling apoptosis in
MCL-1–dependent AML blasts

Downregulating MCL-1 shifts the balance between pro- and anti-apoptotic proteins14

Given the dependence of certain AML blasts on MCL-1, a leading clinical hypothesis posits that downregulating MCL-1 may free pro-apoptotic proteins and their effectors, allowing the apoptotic cascade to be completed.14

MCL-1 is a short-lived protein13

Given the unique vulnerability of MCL-1–dependent AML, clinical efforts have been exploring ways to downregulate MCL-1.16,19This option is attractive for two reasons.

MCL-1 transcription, translation, and degradation are regulated by a large number of pathways13
Physiologic up/down regulation of MCL-1 occurs relatively rapidly due to its short half-life13

Therefore, targeting upstream regulators is expected to reduce MCL-1 levels rapidly. One such regulator is cyclin-dependent kinase 9, or CDK9.13,19

Targeting CDK9 may restore
apoptosis in MCL-1-dependent
AML blasts19

A potential rational treatment strategy for MCL-1–dependent AML

Cyclin-dependent kinase 9, or CDK9, is a crucial upstream regulator of MCL-1.19 CDK9-mediated transcription of MCL-1 may play an important role in the survival of cancer cells, as has been observed in AML and other hematologic malignancies.19,20 Inhibition of CDK9 results in rapid depletion of MCL-1, which may restore apoptosis in AML blasts.13,19

CDK9 as a transcription regulator

CDK9 is a member of the cyclin-dependent kinase (CDK) family of proteins, a group with various important functions for the regulation of cellular processes.21 CDKs are activated by binding to regulatory subunits called cyclins. Most members of the CDK family form a CDK/cyclin complex involved in either cell cycle progression or transcription regulation.21,22
Function22 CDK
Regulation of cell cycle CDK1, CDK2, CDK3, CDK4, CDK6
Regulate transcription and RNA processing CDK7-CDK9 and CDK11-CDK13
CDK9 is a member of the transcription regulating group and does not directly affect cell-cycle control.21 Specifically, CDK9 regulates transcription by phosphorylating a substrate of RNA called carboxyl-terminal domain, or CTD.20 CDK9 is also involved in several other physiologic processes, including differentiation, apoptosis, and DNA repair.19

The mechanics of CDK9

CDK9 generates a heterodimer with regulatory cyclins T1 (CycT), T2a, or T2b to form the main component of the positive transcription elongation factor b (P-TEFb) complex that, when bound to bromodomain protein 4 (BRD4), stimulates transcription elongation by phosphorylating the CTD of the largest subunit of RNA polymerase II. This allows for the expression of genes such as MYC and MCL-1 which may increase the proliferation and survival of cancer cells.19 CDK9 as part of the P-TEFb complex has also been implicated in maintenance of an immature blast phenotype in AML.19
CDK9 with CycT, T2a, or T2b form P-TEFb that when bound to BRD4 stimulates phosphorylation of the CTD of a subunit of RNA polymerase II. Click to expand

CDK9 in cancer

CDK9 dysregulation has been observed in several malignancies

Abnormally elevated CDK9 levels are observable in many cancers. This dysregulation of downstream signaling pathways may result in the abnormal transcription profiles observed in both solid and hematologic tumors, such as19,23:

The onco-mechanism of CDK9

CDK9 activity results in the increased expression and/or hyperactivity of oncogenes such as MCL-1 and MYC. It also promotes blast cell survival mechanisms involving anti-apoptotic factors MCL-1, BCL-2, and XIAP. This is specifically relevant to AML where upregulated MCL-1 gene expression is present in about 50% of relapsed/refractory patients.19

A potential clinical strategy, therefore, is to regulate the expression of CDK9. Additionally, it is hypothesized that inhibition of CDK9 will prevent transcription and may reduce overall levels of mRNA, including MCL-1.19

Inhibition of CDK9 has been intriguing ever since researchers established its connection with oncogenic factors such as MYC, MCL-1, and anti-apoptotic proteins such as BCL-2 and MCL-1.19 The reasoning behind this treatment strategy is strengthened by two factors:

1
A multitude of direct and indirect events such as epigenetic changes, activation of kinase transcription factor, or mutations in regulators may activate CDKs. This provides numerous channels for manipulation, making CDKs notably tractable targets.24
2
Some gene products of CDK9 with oncogenic potential, like MCL-1, have short half-lives, making them sensitive to CDK9 regulation.19

CDK9 targeting in AML

MCL-1 dependency within a subset* of AML blasts provides a therapeutic target.14 The short half-life of MCL-1 means that it is susceptible to the manipulation of upstream regulators.13 Research has shown that CDK9 activity is crucial to maintaining the MCL-1 expression required for MCL-1–dependent AML blast survival.19,21

Inhibition of CDK9 results in reduced levels of MCL-1, reestablishing malignant cells' ability to undergo apoptosis.19,21 Because MCL-1 has a short half-life, the effect is expected to occur rapidly.13

*The prevalence of MCL-1–dependent AML is under investigation.
Watch the effects of CDK9 in
MCL-1–dependent AML.

Preliminary clinical data suggest that CDK9 inhibition may have selective activity against AML blasts, possibly due to dependence on MCL-1. Therefore, MCL-1 regulation via inhibition of CDK9 may be a rational therapeutic strategy for AML.19,23

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