Cyclin-dependent kinase (CDK) 9 and 4/6 inhibitors
in acute myeloid leukemia (AML): a promising
therapeutic approach

Introduction: Despite advancements over the last 2 years, outcomes for acute myeloid leukemia
(AML) are poor; however, a greater comprehension of disease mechanisms has driven the
investigation of new targeted treatments. Cyclin-dependent kinases (CDKs) regulate cell cycle
progression, transcription and DNA repair, and are aberrantly expressed in AML. Targeting the
CDK pathway is an emerging therapeutic strategy with potential.
Areas covered: We describe the rationale for targeting CDK9 and CDK4/6, the ongoing
preclinical and clinical trials and the potential of these inhibitors in AML. Our analysis included
an extensive literature search via the Pubmed database and clinicaltrials.gov (March to August,
Expert opinion: While CDK4/6 inhibitors are early in development for AML, CDK9 inhibition
with alvocidib has encouraging clinical activity in newly diagnosed and relapsed/refractory
AML. Preclinical data suggests that leukemic MCL-1 dependence may predict response to
alvocidib. Moreover, MCL-1 plays a key role in resistance to BCL-2 inhibition with venetoclax.
Investigational strategies of concomitant BCL-2 and CDK9 inhibition is a promising therapeutic
platform for AML. Furthermore, preclinical data suggests that CDK4/6 inhibition has selective
activity in patients with KMT2A-rearrangements and FLT3 mutations. Incorporation of CDK9
and 4/6 inhibitors into the existing therapeutic armamentarium may improve outcomes in AML.
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Acute Myeloid Leukemia, clinical trials, cyclin dependent kinase, CDK9, MCL-1, CDK6,drugdevelopment
Article Highlights
Despite advances in AML, therapeutic options are limited for most patients with
relapsed/refractory disease and outcomes are poor.
Cyclin-dependent kinases (CDK’s) regulate cell cycle progression, transcription and
DNA repair and inhibition of CDK’s represents a promising investigational approach in
CDK9 inhibition down-regulates the transcription of genes such as c-MYC and MCL-1
that are involved in cell survival
CDK6 inhibition leads to cell cycle arrest in G1 phase and is a promising therapeutic
approach in KMT2A (MLL)-rearranged and FLT3-mutated AML
Alvocidib (flavopiridol), a CDK9 inhibitor, has shown the most promise as a CDK
inhibitor in AML
Alvocidib has demonstrated clinical activity when combined with cytarabine and
mitoxantrone (FLAM) in newly diagnosed and relapsed/refractory AML.
Biomarker-based strategies to identify patients most likely to benefit from CDK
inhibition in AML is a future challenge
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1. Introduction
Acute myeloid leukemia (AML) is an aggressive malignancy of immature myeloid progenitor
cells. Approximately 21,000 people will be diagnosed with AML in the United States each year,
and nearly 11,000 will die from the disease [1]. The incidence of AML increases with age, and
the median age at diagnosis is approximately 65 years old [2, 3]. There have been several recent
therapeutic advances and drug approvals by the Food and Drug Administration (FDA) for AML,
including CPX-351, a liposomal formulation of daunorubicin and cytarabine, glasdegib, a small
molecule inhibitor of the Hedgehog pathway, and targeted therapies against FLT3- (midostaurin
and gilteritinib), IDH1- (ivosidenib), and IDH2- (enasidenib) mutated AML [4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16]. The FDA also granted accelerated approval to venetoclax (ABT-199), a
BCL-2 inhibitor, combined with hypomethylating agents (HMAs) or low-dose cytarabine
(LDAC) for elderly AML patients (>75 years) or those who are not candidates for intensive
chemotherapy based on results from two single-arm phase 1b studies revealing an impressive
complete remission/complete remission with incomplete count recovery (CR/CRi) rate of 67%
and 54% and median OS of 17.5 months and 13.5 months in combination with either HMAs or
LDAC, respectively [17, 18, 19]. Randomized studies are ongoing comparing azacitidine plus
venetoclax to azacitidine alone (NCT02993523) and LDAC plus venetoclax versus LDAC alone
for newly diagnosed elderly AML patients (NCT03069352). However, many of these new
therapies are indicated only for certain AML subsets, and the improvements in response and
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median OS are modest. Novel approaches and treatments are needed that are potentially more
generalizable or inhibit resistance pathways.
The cyclin-dependent kinases (CDKs) are promising therapeutic targets under
investigation in AML. CDKs combine with cyclin regulatory subunits to form complexes that
control cell cycle progression, transcription, DNA repair, epigenetic regulation, and other
important processes involved in proliferation, differentiation, and apoptosis [20, 21].
Dysregulation of CDK-controlled pathways has been implicated in several tumor subtypes,
including AML [22, 23]. This review will focus on CDK9 and CDK4/6 inhibitors currently in
clinical development (see tables 1, 2 and 3). Our analysis included an extensive review of the
published literature of CDK9 and CDK4/6 inhibitors in AML via the Pubmed database as well as
a search of ongoing clinical trials via the ClinicalTrials.gov database from March-August, 2019.
Their mechanisms of action and roles in AML will be briefly reviewed, and the clinical
experience to date with CDK9 and CDK4/6 inhibitors will be summarized.
2. CDK9 and Its Role in AML
CDK9 regulates transcriptional elongation by forming a complex with regulatory cyclin T1
known as positive transcription elongation factor b (P-TEFb).[24, 25] P-TEFb exists in a super￾enhancer complex and phosphorylates RNA polymerase II, which thereby activates global
transcription elongation of important genes involved in cell proliferation and survival such as
MYC and MCL-1 (Figure 1) [26, 27]. MYC and MCL-1 are up-regulated and implicated in cell
survival for a multitude of tumor subtypes. Thus, reduction of transcription of these pro-survival
genes is one of the primary rationales for inhibiting CDK9 in cancer.
CDK9-mediated pathways have been implicated in the development and survival of
leukemic cells in AML, and research has focused on BCL-2 family member MCL-1. MCL-1 was
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isolated from the ML-1 human myeloid leukemia cell line and was found to have sequence
similarity to BCL-2 [28]. MCL-1, like other antiapoptotic BCL-2 family members, sequesters
BH3 proapoptotic proteins such as BIM, NOXA and PUMA, thereby inhibiting the BAX and
BAK-mediated intrinsic pathway of apoptosis [29, 30] (Figure 1). MCL-1 has a very short half￾life (2-4 hours), denoting its dependence on transcriptional activation and limiting the therapeutic
targeted inhibition of MCL-1 [31]. Overexpression of MCL-1 delayed apoptosis in
hematopoietic cells, including myeloid leukemia cells [32, 33]. Conditional deletion of MCL-1
caused apoptosis of leukemia cells in mouse models of AML, as did functional inactivation of
MCL-1 in human leukemia-derived cell lines and primary patient samples, suggesting a critical
role for MCL-1 in AML survival [34]. MCL-1 has been found to be highly expressed in de novo
primary AML samples, and it is also frequently overexpressed in relapsed/refractory AML [35,
Targeting BCL-2 family proteins to promote apoptosis is a promising approach in AML.
Venetoclax combined with hypomethylating agents produces encouraging response rates [18].
However, venetoclax resistance as well as relapse after CR represent significant limitations of
this agent. MCL-1 is stabilized and levels are increased in venetoclax-resistant AML cell lines.
BIM binds to MCL-1 in these cell lines and is one pathway for venetoclax resistance [37]. These
findings indicate that CDK9-mediated suppression of MCL-1 is an attractive target for drug
development in AML.
3. CDK9 Inhibitors in Clinical Development for AML
3.1 Alvocidib
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Alvocidib, previously known as flavopiridol, is a multi-CDK inhibitor that potently inhibits
CDK9 (Kd = 6) [38]. Originally, alvocidib’s mechanism of action was attributed to its arrest of
the cell cycle at the G1 phase via CDK4/6 blockade [39, 40]. However, it is now understood that
the primary mechanism of action of alvocidib is transcriptional regulation through CDK9/P￾TEFb inhibition, independent of its inhibition of the cell cycle, which has been corroborated by
preclinical studies in multiple hematologic malignancy models [41, 42, 43, 44, 45, 46, 47, 48, 49,
50]. This CDK9 inhibition downregulates transcripts critical for survival and proliferation of
cancer cells, such as cyclin D1, c-MYC, and importantly MCL-1.
The integration of alvocidib into a timed sequential cytotoxic induction regimen for AML
patients was originally conceived by Dr. Judith Karp and colleagues. Notably, an in vitro timed
sequential therapy model of relapsed/refractory AML patient samples revealed a 4.3-fold
increase in apoptosis and a significant increase in cytotoxicity with the addition of alvocidib
followed by cytarabine versus either alvocidib or cytarabine alone [46]. These results revealed
that alvocidib may potentiate the effects of cytotoxic chemotherapy in AML patients.
This pivotal analysis formed the basis of a phase I dose escalation study of intravenous
(IV) bolus alvocidib (days 1-3) followed by timed sequential cytarabine (667 mg/m2
continuous infusion IV days 6-8) and mitoxantrone (40 mg/m2
IV day 9), known as the FLAM
regimen. Thirty-four adults with poor-risk, relapsed, or refractory AML (n = 26), acute
lymphoblastic leukemia (ALL, n = 7), or blast crisis chronic myeloid leukemia (CML, n = 1)
were enrolled on this phase I study. The maximum tolerated dose (MTD) and recommended
phase II dose (RP2D) of IV bolus alvocidib was determined to be 50 mg/m2
/day. Significant
toxicities included tumor lysis syndrome (26%), grade 3 diarrhea (9%), and grade 2 or higher
mucositis (12%). The complete response (CR) rate was 21%, and the partial response (PR) rate
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was 6%, for an overall response rate (ORR) of 27%. In AML patients, the ORR was 31%, with a
CR rate of 23% and a PR rate of 8%. Pharmacodynamic studies confirmed on-target decreases in
cyclin D1, BCL-2, MCL-1, and phosphorylated RNA polymerase 2 [51].
Subsequently, the RP2D of bolus alvocidib was studied in a phase II trial of 62 adults
with relapsed (n =24), refractory (n = 23), and untreated poor-risk secondary (n = 15) AML. All
patients received IV bolus alvocidib at 50 mg/m2
/day on days 1-3 followed by the same timed
sequential backbone of cytarabine and mitoxantrone (FLAM) as the earlier phase I. Adverse
events were similar to the prior phase I, with mucositis, diarrhea, and tumor lysis syndrome
being the most common significant toxicities. Impressive clinical activity was seen, with CR
rates of 75%, 75%, and 9% for untreated secondary AML, relapsed AML, and refractory AML
participants, respectively. Of the 32 patients who achieved CRs, 12 went on to undergo
allogeneic stem cell transplantation. Median overall survival (OS) for all of the participants was
8 months, and median disease free survival (DFS) for those who achieved CR was 11 months
Based on these promising results, a follow up phase II study was conducted in 45 adults
with newly diagnosed poor-risk AML including secondary AML and unfavorable-risk
cytogenetics. Bolus alvocidib within the FLAM regimen was administered identically to the
prior phase II study. Adverse events included tumor lysis syndrome, oral mucositis, and diarrhea,
but cardiac dysfunction was also notable (16%). Thirty- and 60-day treatment-related mortality
were 4% and 9%, respectively. The overall CR rate was 67%, and the median OS was 7.4
months. For those subjects that achieved CR, 40% underwent allogeneic stem cell
transplantation, and the median DFS was 13.3 months [53].
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These results led to a randomized phase II study of FLAM versus 7+3 (cytarabine 100
/day continuous infusion IV days 1-7, daunorubicin 90 mg/m2
IV days 1-3) in newly
diagnosed AML with intermediate- or poor-risk cytogenetics. Reinduction with 5+2 (cytarabine
100 mg/m2
/day days 1-5 with daunorubicin 45 mg/m2
days 1-2) was permitted for residual
leukemia noted on day 14 bone marrow for the 7+3 treatment arm, but reinduction was not given
to patients on the FLAM arm. A total of 165 patients were enrolled and randomized in a 2:1
fashion to receive FLAM (n=109) versus 7+3 (n=56).[54] Per the 2010 European LeukemiaNet
(ELN) classification, 44% and 38% were adverse-risk in the FLAM and 7+3 arms, respectively
[54, 55]. Additionally, 48% enrolled into the FLAM arm had secondary AML versus 46% in the
7+3 arm. Toxicities were similar between the two arms, but 60-day mortality was non￾significantly higher in the FLAM arm as compared to the 7+3 arm (10% vs 4%, p = 0.22). Most
of those early deaths with FLAM were in participants ≥60 years of age. The CR rate was
significantly increased in the FLAM arm compared with 7+3 arm (70% vs. 46%, p = 0.003).
When compared to those participants who received reinduction with 5+2 for residual leukemia
on the 7+3 arm, the CR rate remained higher with FLAM (70% vs. 57%, p = 0.08). Post￾remission therapy varied per investigator preference and was not controlled. Rates of allogeneic
stem cell transplant were similar between the two arms (51% FLAM vs 48% 7+3). Furthermore,
there was no significant difference in median OS between FLAM and 7+3 (17.5 months vs 23.1
months, p = 0.5) despite a non-significant improvement in event-free survival with FLAM (9.7
months vs. 3.4 months, p=0.18). Subset analyses revealed a significant improvement in CR and a
non-significant improvement in OS in younger patients (<50 years) treated with FLAM versus
7+3 [56].
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Significant protein binding of alvocidib in human serum was discovered in vitro, so a
“hybrid” treatment schedule was developed to maximize drug exposure. This hybrid schedule,
where 1/3 to 1/2 of the total dose of alvocidib is given as a 30-minute bolus followed by a 4-hour
infusion, was first tested as monotherapy in refractory CLL with promising results [57]. The
hybrid alvocidib treatment was subsequently investigated as a single agent in acute leukemia in a
phase I dose escalation study. Twenty-four adults, 19 with relapsed/refractory AML and 5 with
relapsed/refractory ALL, were given alvocidib via a 30-minute bolus followed by a 4-hour
infusion for three days. The alvocidib dose started at 20 mg/m2
bolus with 30 mg/m2
and reached 50 mg/m2
bolus with 75 mg/m2
infusions before reaching dose-limiting diarrhea.
One AML subject achieved a CR with incomplete hematologic recovery (CRi) [58].
Given that single agent alvocidib did not demonstrate substantial clinical activity, a phase
I trial was conducted to determine the MTD and RP2D of hybrid alvocidib when given with the
same timed sequential cytarabine and mitoxantrone regimen that was studied with bolus
alvocidib (FLAM). Fifty-five adults with relapsed/refractory AML (n = 49), ALL (n = 3), and
biphenotypic leukemia (n = 3) were enrolled. Tumor lysis syndrome was observed in 51% of
participants, and significant cardiac toxicities occurred in 9% of subjects. The MTD and RP2D
of alvocidib was determined to be 30 mg/m2
bolus with 60 mg/m2
infusions. The overall CR rate
was 40%, and an additional 5% achieved PR, for an ORR of 45%. The CR rate at the RP2D was
52%. Of the participants who were in CR, 73% underwent allogeneic stem cell transplant.
Median OS was 7.4 months, and median DFS was not reached (range 1.8 to 30 months) at the
time of publication [59].
A randomized phase II study was then undertaken by Karp et al [60]. comparing hybrid
versus bolus formulations of alvocidib within the FLAM regimen in newly diagnosed poor-risk
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AML (>50 years, secondary AML, and/or adverse-risk cytogenetics). The hybrid alvocidib dose
chosen for this study was 30 mg/m2
IV bolus followed by 40 mg/m2
IV over 4 hours due to
tumor lysis syndrome, mucositis, diarrhea and hyperbilirubinemia seen with hybrid alvocidib
despite the MTD of 30 mg/m2
IV bolus followed by 60 mg/m2
IV over 4 hours seen in the prior
phase I study. This study enrolled a highly poor-risk subset of patients as most patients enrolled
on this study had either secondary AML or adverse-risk AML. Toxicities and treatment-related
mortality was similar between bolus and hybrid FLAM. CR rates were not significantly different
between bolus FLAM and hybrid FLAM (62% vs. 74%) with promising results seen in
secondary AML (65% vs. 71%) and adverse-risk cytogenetics (68% vs. 67%). Median OS was
11.4 months versus 13.0 months with bolus versus hybrid FLAM, respectively (p=0.38) [60].
As previously discussed, one important mechanism for alvocidib’s anti-tumor activity
appears to be transcriptional downregulation of MCL-1 through CDK9 inhibition. In an effort to
assess for a predictive biomarker for response to alvocidib, mitochondrial BH3 profiling was
done on 63 archived samples from the randomized phase II study of bolus FLAM versus 7+3
[54, 61]. BH3 profiling is an analysis of tumor dependence on anti-apoptotic proteins and
measures apoptotic sensitivity or “priming” to selective pro-apoptotic BH3 peptides [62, 63]. For
example, NOXA is a BH3 pro-apoptotic protein that selectively antagonizes MCL-1. Tumors
exposed to NOXA peptides that lead to a high degree of apoptosis (i.e. NOXA priming) suggest
that the tumor is dependent on MCL-1 for cell survival [64]. NOXA priming was significantly
higher in the bone marrow samples from AML patients who achieved CR with FLAM as
compared to non-responders (44.5% primed vs 5.2% primed, respectively, p = 0.006). None of
the non-responders had NOXA priming higher than 40% in bone marrow samples [61]. Thus,
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BH3 profiling from the bone marrow to assess for MCL-1 dependence represents an attractive
strategy to predict sensitivity to alvocidib containing regimens in AML.
A phase II biomarker-driven study (Zella-201) is ongoing to determine whether MCL-1
dependence can be used to predict response to alvocidib (NCT02520011). This study is enrolling
MCL-1 dependent relapsed/refractory AML patients with NOXA priming ≥40% in two stages.
In Stage 1, all participants receive hybrid FLAM (alvocidib 30 mg/m2
IV bolus followed by 60
IV over 4 hours on days 1-3, cytarabine 667 mg/m2
/day continuous infusion days 6-8,
mitoxantrone 40 mg/m2
IV day 9). If >13 CRs are observed in the first 23 patients enrolled in
Stage 1, then patients will be randomized to receive hybrid FLAM or cytarabine and
mitoxantrone (AM) in Stage 2. The primary endpoint of the study is the rate of CR + CRi. Stage
1 results were presented at the 2018 American Society of Hematology (ASH) Annual Meeting.
Of 170 relapsed/refractory AML patients screened, 48 (28%) patients were found to be MCL-1
dependent. The CR/CRi rate among the overall patient population and those evaluable for
response was 57% (13/23) and 68% (13/19), respectively. Notably, 58% of refractory AML
patients achieved a CR/CRi in Stage 1. Overall toxicities seen in Stage 1 were consistent with
previously published results with FLAM. Stage 2 of the study is ongoing to determine whether
the addition of alvocidib to cytotoxic chemotherapy improves CR rates in MCL-1 dependent
relapsed/refractory AML patients [65].
Finally, hybrid alvocidib is being studied in combination with standard 7+3
chemotherapy in a dose escalation phase I trial (Zella-101) in newly diagnosed AML with non￾favorable cytogenetics (NCT03298984). The primary endpoint is the MTD and RP2D of hybrid
alvocidib when administered with cytarabine 100 mg/m2
days 1-7 and daunorubicin 60 mg/m2
days 1-3. Preliminary results were presented at the 24th European Hematology Association
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(EHA) Congress in 2019. Eighteen patients were enrolled and the MTD and RP2D of alvocidib
was determined to be 30 mg/m2
over a 30-minute bolus followed by 60 mg/m2
over 4 hours days
1-3, consistent with the MTD seen in combination with timed sequential cytarabine and
mitoxantrone (FLAM). Toxicities were primarily infections related to AML and cytotoxic
induction chemotherapy, but diarrhea and tumor lysis syndrome were the most common drug￾related adverse events. Fourteen of the 18 patients (78%) achieved CR/CRi to date. An
expansion cohort at the RP2D is ongoing, and correlative BH3 profiling studies are being
performed [66].
Targeting complementary BCL-2 family proteins, such as MCL-1, could enhance
venetoclax activity or inhibit resistance pathways. In a preclinical study, Bogenberger et al
demonstrated that venetoclax combined with alvocidib was synergistic in both venetoclax￾sensitive and venetoclax-resistant AML models and primary patient samples. This synergy was
mediated through CDK9 inhibition, which decreased MCL-1 and increased levels of pro￾apoptotic proteins, leading to increased apoptosis over either agent alone [67]. Clinical trials are
ongoing investigating the combination of alvocidib and venetoclax in relapsed/refractory AML
(NCT03441555) and alvocidib after venetoclax failure in AML (NCT03969420).
A recent analysis of 154 newly diagnosed AML patients identified three biologically
distinct refractory AML subgroups based on RNA-sequencing transcriptome analysis. Ex vivo
drug sensitivity was conducted with >100 small molecule inhibitors in each of these refractory
AML subgroups. Notably, alvocidib had a more robust cytotoxic effect in refractory AML
patient samples and those with adverse-risk disease when compared with those who achieved CR
and favorable-risk disease, respectively. Moreover, alvocidib had the strongest clinical activity in
a refractory subgroup with increased expression of pathways involved in the cell cycle and DNA
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replication/repair [68]. These findings substantiate alvocidib’s anti-leukemic activity in
refractory AML patients and continue to make a compelling case for innovative biomarker-based
study designs of alvocidib in AML.
3.2 Dinaciclib
Dinaciclib (SCH-727965) is a more selective CDK inhibitor than alvocidib with activity against
CDK1, CDK2, CDK5, and CDK9 [69]. It was originally selected for development due to its
balance of activity and tolerability in an ovarian cancer xenograft screening system. In this
model, dinaciclib was more potent and tolerable than alvocidib [70]. Dinaciclib inhibited Rb
phosphorylation in vitro, which correlated to induction of apoptosis, and demonstrated
antiproliferative effects in >100 tumor cell lines, including leukemia cell lines, at low nanomolar
IC50 [69].
In a small-molecule inhibitor screen in KMT2A (also known as MLL)-rearranged AML
cells, dinaciclib was found to be one of the most potent apoptosis inducers in the screen.
Treatment of these KMT2A-driven AML cells with dinaciclib led to rapid decreases in MCL-1,
and MCL-1 overexpression protected the AML cells from apoptosis. Potent antitumor responses
were seen in mouse models of KMT2A-rearranged acute leukemia [71].
A randomized phase II study of dinaciclib versus gemtuzumab ozogamicin, a CD33
antibody-drug conjugate, in relapsed/refractory AML was conducted, and the experience with
dinaciclib was reported [72]. Dinaciclib was given at 50 mg/m2
IV over 2 hours on day 1 of
cycle 1, and intra-patient dose escalation was permitted to 70 mg/m2
IV over 2 hours on day 1 of
each subsequent 21-day cycle based on an earlier phase I dose-escalation study in multiple
advanced cancers [73]. Fourteen patients with AML received dinaciclib on this study. Toxicities
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were frequent, including diarrhea and grade 3 or higher fatigue. Half of the participants
developed tumor lysis syndrome, and one subject died of hyperacute tumor lysis that led to acute
renal failure. No objective responses were observed. Pharmacodynamic studies from 1 AML and
3 ALL patients treated with dinaciclib showed decreases in MCL-1 in all specimens at 4 hours
post-treatment, but these decreases were short-lived, and MCL-1 expression returned to baseline
by 24 hours. Decreased phosphorylated Rb protein was also observed in 1 participant, but 2
subjects had nearly undetectable phosphorylated Rb pre-treatment. Additional in vitro studies
demonstrated improved MCL-1 downregulation and apoptosis of primary AML samples with
prolonged dinaciclib exposure [72].
Combined venetoclax and dinaciclib displayed potent synergy in xenograft and mouse
models of diffuse large B-cell lymphoma [74]. This potential synergy is being studied in a phase
I dose-escalation study of venetoclax and dinaciclib in relapsed/refractory AML
3.3 Atuveciclib and BAY 1251152
Atuveciclib (BAY 1143572) is an oral CDK inhibitor that potently and selectively inhibits
CDK9/PTEFb. The IC50 of atuveciclib for CDK9 is 13 nmol, while its inhibitory activities
against the other CDKs are in the micromolar range. Atuveciclib demonstrated antitumor activity
in xenograft mouse and rat models through inhibition of phosphorylation of RNA polymerase II
and downregulation of c-MYC and MCL-1 [75, 76, 77]. In AML cell lines, including KMT2A￾rearranged lines, atuveciclib induced apoptosis with a median IC50 of 385 nM, and also showed
potent activity in primary AML samples [76]. Daily oral administration of atuveciclib led to
partial and complete remissions in AML xenograft models in mice and rats [76, 77].
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Based off these preclinical studies, a phase I dose escalation study was conducted to
determine the MTD and RP2D of atuveciclib monotherapy in patients with relapsed/refractory
acute leukemias (NCT02345382). This study has been completed, but no results have been
BAY 1251152 was later identified as an attractive CDK9 inhibitor based on the
preclinical and early phase clinical trials with atuveciclib. BAY 11251152 is an IV CDK
inhibitor that has increased activity against CDK9 (IC50 = 3nM) and CDK2 when compared to
atuveciclib, and it demonstrated efficacy in AML xenograft models [78].
A phase I dose escalation study of BAY 1251152 was completed, and the results were
presented at the 2018 ASH Annual Meeting. Thirty patients with relapsed or refractory AML
were enrolled, and 21 participants were treated. BAY 1251152 was administered IV over 30
minutes once weekly for 21-day cycles. Four dose levels were tested (5, 10, 20, and 30mg), and
an MTD was not defined. Significant adverse events were primarily hematologic. No objective
responses were observed. Pharmacodynamic studies indicated that treatment with BAY 1251152
led to significant but temporary reductions in c-MYC and MCL-1 [79].
3.4 TG02
TG02 is an oral inhibitor with activity against CDKs 1, 2, 7, and 9, as well as JAK2 and FLT3.
The IC50 for CDK9 is 3 nM, while the IC50 for JAK2, FLT3, and FLT3-D835 are 19 nM, 19 nM,
and 21 nM, respectively. TG02 potently inhibited proliferation of several AML cell lines, and
on-target RNA polymerase II effect was observed, leading to reduced levels of pro-survival
proteins such as MCL-1 [80, 81]. Complete tumor regression was obtained in AML mouse
models with TG02 therapy [80]. Additionally, using dynamic BH3 profiling, TG02 was shown to
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sensitize AML cells to BCL-2-inhibitory BAD-BH3 peptide, while venetoclax sensitized AML
cells to MCL-1-inhibitory NOXA-BH3 peptide, suggesting a mechanism for synergy. Increased
apoptosis was observed with combination TG02 and venetoclax therapy in AML cell lines and
primary patient samples over treatment with each agent individually [82].
A phase I dose escalation study of TG02 in advanced acute leukemias and relapsed
multiple myeloma has been completed (NCT01204164), but results for the leukemia participants
have not been reported.

4. CDK6 and Its Role in AML
CDKs 4 and 6, in combination with their activating cyclins D1, D2, or D3, control progression
through the G1-S phase transition by phosphorylating the retinoblastoma (Rb) protein [83, 84,
85]. Hypophosphorylated Rb normally binds to and inhibits the activity of the E2F family of
transcription factors [86, 87]. Phosphorylation of Rb by CDK4/6 then relieves its inhibition of
E2F, allowing transcription of genes needed for DNA synthesis and S phase transition to proceed
(Figure 2) [88, 89].
CDK6 is preferentially expressed in hematopoietic cells and is involved in hematopoiesis
[90, 91, 92]. CDK6 knockout mice had fewer red blood cells, megakaryocytes, neutrophils, and
macrophages when compared to CDK6 wildtype mice, and CDK6 deficient lymphocytes had
delayed S phase entry [92]. CDK6 activity has been implicated in KMT2A-rearranged AML [93].
One function of aberrant KMT2A rearrangements is transforming hematopoietic stem and
progenitor cells to leukemia [94, 95]. Placke et al demonstrated that rearranged KMT2A binds to
the CDK6 locus and that increased expression of CDK6 but not CDK4 drives AML in KMT2A￾rearranged AML. Further, KMT2A-rearranged AML relies on CDK6 to maintain immaturity, as
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knockdown or pharmacologic inhibition of CDK6 resulted in decreased growth but also
differentiation of the leukemia cells [93].
CDK6 activity has also been linked to FLT3-ITD mutated AML [96, 97]. In FLT3-ITD
AML cell lines, inhibition of the FLT3-ITD kinase led to decreased expression of cyclins D2 and
D3, with a concurrent decrease in phosphorylated Rb levels, suggestive of decreased CDK4/6
activity [96]. Inhibition of FLT3-ITD also has been shown to decrease CDK6 expression through
the SRC-family of kinases [97]. These data offer rationales for targeting CDK6 in two poor risk
subtypes of AML (i.e. KMT2A-rearrangements and FLT3-ITD mutations).
5. CDK4/6 Inhibitors in Clinical Development for AML
5.1 Palbociclib
Palbociclib (PD-0332991) is an oral CDK4/6 inhibitor currently approved for the treatment of
hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative
advanced or metastatic breast cancer in combination with an aromatase inhibitor or with
fulvestrant. Palbociclib potently and specifically inhibits CDK4 (IC50 11 nmol/L) and CDK6
(IC50 15 nmol/L) [98]. Treatment of AML cell lines and primary patient samples with palbociclib
leads to significant G1 phase arrest, which sensitizes AML cells to cytarabine cytotoxicity, as
release of the CDK4/6 inhibition leads to the transition of AML cells into S phase of the cell
cycle [96, 99]. In KMT2A-rearranged AML cell lines and patient samples, which rely on CDK6
activity to maintain an immature phenotype, palbociclib therapy prohibited growth and induced
differentiation [93]. Finally, palbociclib causes cell-cycle arrest, downregulates FLT3
transcription, and synergizes with FLT3 inhibitors in preclinical models of FLT3-ITD-mutated
AML [96, 100].
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A phase Ib/IIa trial studying palbociclib monotherapy in KMT2A-mutated acute leukemia
(NCT02310243) reported interim results at the 2016 ASH Annual Meeting. Palbociclib was
administered at 125 mg once daily for 21 days of 28-day cycles to six relapsed/refractory acute
leukemia participants. Five subjects had AML, while one had acute lymphoblastic leukemia. No
DLTs were observed. One participant achieved a CR with incomplete hematologic recovery after
two cycles of therapy, three had stable disease, and two progressed [101]. The expansion phase
of this study as well as combination trials (NCT03844997, NCT03132454) are ongoing.
5.2 FLX925 is an oral, rationally designed FLT3 and CDK4/6 inhibitor that was
developed to address FLT3 resistance mechanisms. FLX925 demonstrated an improved
resistance profile when compared FLT3 inhibitors quizartinib and sorafenib in FLT3-ITD
mutated AML cell lines, and FLX925 treatment strongly suppressed proliferation of a panel of
AML cell lines, including FLT3-wildtype lines [102].
A phase I study with FLX925 was completed, and the results of the trial were reported at
the 2017 ASH Annual Meeting. FLX925 monotherapy was administered orally two to three
times daily to 51 relapsed/refractory AML participants. Both FLT3-mutated and -wildtype
patients were enrolled and treated. The most common treatment-related adverse events were
nausea, diarrhea, increased creatinine, and acute or chronic renal failure. Two DLTs were
observed at 720 mg three times daily, and both were due to kidney dysfunction. The MTD was
determined to be 540 mg three times daily. Two of the 21 patients achieved transient
morphologic leukemia-free states (both FLT3-mutated), but no objective responses were
obtained [103].
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6. Conclusions
Dysregulation of pathways controlled by the CDKs is a key feature of many malignancies. In
AML, CDK9 and CDK6 affect critical processes involved in tumorigenesis, maintaining
immaturity, proliferation, and potential resistance pathways to currently available therapies.
Inhibiting CDK9 or CDK6 is a novel approach to treating AML, especially in rational cytotoxic
chemotherapy or target therapy combinations. Targeting the resistance mechanisms to BCL-2
and FLT3 inhibitors with CDK9 and CDK6 inhibitors is particularly intriguing. Several CDK9
and CDK4/6 inhibitors are currently in clinical development and alvocidib has shown promising
efficacy in relapsed/refractory and treatment naïve AML. Multiple studies are ongoing to assess
the role of these agents in AML.
7. Expert Opinion
Our understanding of the genomic complexity of AML has evolved over the last several years.
The largest genomic classification of AML was recently reported whereby somatic driver
mutations were assessed in 1,540 newly diagnosed AML patients who enrolled on three
prospective multicenter clinical trials through the German-Austrian AML Study Group. This
analysis revealed 11 distinct genomic subgroups with biologic and prognostic significance,
highlighting the heterogeneity of AML and the unique attributes of each individual tumor [104].
The most common mutations observed in AML are NPM1 and FLT3 mutations, each seen in
approximately 25-30% of patients with substantial overlap. The majority of AML patients have
at least one driver mutation that is identifiable through next generation sequencing (NGS), and
multiple subclones can emerge throughout treatment that can be intrinsically chemoresistant.
Subclassification of AML into distinct genomic subsets will provide a springboard for the
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development of novel targeted therapies allowing us to move closer to a more personalized
therapeutic approach for AML.
We are now transitioning beyond a one-size fits all therapeutic approach in AML (i.e.
7+3) which has been the cornerstone of treatment for newly diagnosed AML patients for the last
4 decades. All AML patients should have testing for cytogenetics and mutational status through a
targeted NGS panel for therapeutic and prognostic purposes. The optimal induction
chemotherapy regimen selected for patients should be based on individual risk stratification and
biologic features. For example, CPX-351 (liposomal cytarabine and daunorubicin) leads to
significant improvement in OS when compared with 7+3 in patients with AML with MDS￾related changes and represents a new standard of care for this patient population [5].
Gemtuzumab ozogamicin (GO), a monoclonal antibody targeting CD33 with a DNA-damaging
linker (calicheamicin), has been shown to significantly improve event-free survival (EFS) when
combined with 7+3 compared with 7+3 alone. Subset analyses from this randomized phase 3
study revealed that only favorable and intermediate-risk patients benefit from the addition of GO
and should thus be considered for patients with favorable and intermediate-risk subtypes but not
those with adverse-risk disease [105, 106]. Finally, the addition of midostaurin, a multi-kinase
FLT3 inhibitor, has been shown to prolong OS when combined with 7+3 in patients with FLT3
mutations [9]. Although targeted therapeutic approaches (i.e. midostaurin and gilteritinib for
FLT3 mutations, ivosidenib and enasidenib for IDH1 and IDH2 mutations, respectively) are
becoming a mainstay of our therapeutic armamentarium for AML, the majority of patients
treated with single-agent mutation-directed therapies do not respond, durable CRs are extremely
rare, and long-term outcomes remain poor. While development of these mutation-specific
therapies are important, we believe that drug development in the future will need to rely on the
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identification of predictive signatures of response (and resistance) to selected targeted agents
without relying solely on molecular abnormalities. Inclusion criteria of select targeted
combination approaches should be increasingly narrow and based on biologically plausible
mechanisms of response in order to ultimately tailor therapeutic approaches to patients most
likely to achieve long-term response.
The recent accelerated approval of venetoclax (BCL-2 inhibitor) in combination with
HMAs or LDAC in newly diagnosed elderly, unfit AML has led to a paradigm shift in the front￾line management of elderly AML. Although randomized phase 3 studies are still ongoing
investigating whether venetoclax plus azacitidine or venetoclax plus LDAC improves OS when
compared with azacitidine or LDAC alone, respectively, the high response rates associated with
venetoclax combinations in the upfront setting has renewed optimism for this poor-risk group of
patients with median OS traditionally being 6-12 months [107, 108, 109]. Venetoclax plus
HMAs or LDAC represents a new standard-of-care for elderly AML patients and is widely used
in the front-line setting for this patient population. Given the promising clinical activity seen
with venetoclax-based combinations in newly diagnosed elderly AML, venetoclax is being
investigated in combination with other front-line treatment approaches such as induction
chemotherapy for newly diagnosed, younger fit AML patients (NCT03709758, NCT03629171,
NCT03455504, NCT03214562, NCT02115295, NCT04075747), and is likely to be further
incorporated into diverse treatment backbones in AML. Unfortunately, response to venetoclax￾based strategies is not durable. Predictive genomic signatures and biomarkers predicting
heightened sensitivity to venetoclax is sorely needed in AML in order to abrogate unnecessary
toxicity in patients unlikely to respond to this agent.
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BH3 profiling represents an attractive and promising platform to determine whether
leukemic cells are dependent on BCL-2, BCL-XL or MCL-1 for survival [110]. This strategy
would allow a personalized based treatment approach to target pro-survival mechanisms of
leukemic cells. Zella-201 is a biomarker-based randomized phase 2 study comparing alvocidib,
cytarabine and mitoxantrone (FLAM) versus cytarabine and mitoxantrone (AM) in MCL-1
dependent AML patients. The stage 1 CR/CRi rate was impressively high in this high-risk
patient population, especially for those with primary refractory AML [65]. Clinical trials
incorporating BH3 profiling as a predictor of response to BCL-2 and MCL-1 inhibitors are
imperative to further our understanding of mechanisms of response and resistance to these agents
in AML. This strategy represents the quintessence of targeted therapy for individual patients.
We believe that rational biomarker-driven studies that incorporate cell survival signals,
molecular abnormalities, and other genomic features that preferentially select for responders to
distinct regimens will continue to move the field forward.
Multiple studies have shown that up-regulation of MCL-1 is a dominant resistance
mechanism in patients treated with venetoclax [37, 67, 111, 112, 113]. Suppression of MCL-1
via CDK9 inhibition could boost or rescue sensitivity to BCL-2 inhibitors such as venetoclax.
This is an important area of interest due to the widespread use of venetoclax combinations in
AML. Several studies are ongoing with CDK9 inihibitors (alvocidib and dinaciclib) in
combination with venetoclax to evaluate whether the potent pre-clinical synergy seen with BCL-
2 and MCL-1 inhibition translates into better outcomes in patients and can potentially mitigate
resistance seen with BCL-2 inhibition. Pre-clinical and correlative studies are critical to
determine whether there may be increased synergy with concomitant BCL-2 and MCL-1
inhibition versus a sequential therapy (i.e. BCL-2 inhibition followed by MCL-1 inhibition)
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approach in AML. A multicenter randomized phase 2 study is evaluating whether alvocidib
treatment (with or without LDAC) after venetoclax failure (NCT03969420) is effective in AML
with the hypothesis that MCL-1 may be driving resistance and relapse after venetoclax-based
therapies. The results of these ongoing trials will serve as a linchpin for future BCL-2 and MCL-
1 inhibitory strategies. Similarly, combined FLT3 and CDK6 inhibition is also being tested to
address FLT3 inhibitor resistance, which remains a significant problem for patients treated with
FLT3 inhibitors such as gilteritinib.
Given the clonal heterogeneity of AML, it is unlikely that single-agent therapy will
provide durable responses. We believe that combinatorial approaches should be studied earlier in
drug development to fully elucidate the clinical activity of selected investigational agents and to
target potential resistance pathways that inevitably occur with single-agent therapies. CDK9 and
4/6 inhibitors show promise for incorporation into rational combinations in the frontline setting
as well as in relapsed/refractory disease. We are optimistic that the incorporation of these novel
agents into our existing treatment armamentarium for specific subpopulations of AML predicted
to respond to CDK9 and 4/6 inhibition will lead to improved clinical outcomes and come closer
to our unified goal of curing AML.
This paper was not funded.
Declaration of interest
JF Zeidner has received honoraria from AbbVie, Agios, Celgene, Daiichi Sankyo, Pfizer, and
Tolero. He has also served as a Consultant for AsystBio Laboratories, and Celgene and has
received research funding from Celgene, Merck, Takeda, and Tolero.
DJ Lee has served as a Consultant for Boston Biomedical, and has received research funding
from Abbvie, Bayer, F. Hoffman-La Roche, Forty Seven, and Tolero. The authors have no other
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relevant affiliations or financial involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose
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Information Classification: General
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Accepted Manuscript
Information Classification: General
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Accepted Manuscript
Information Classification: Genera
Abbreviations: MTD – maximum tolerated dose; AML – acute myeloid leukemia; ALL – acute
lymphoblastic leukemia; CR – complete remission; FLAM – flavopridol, cytarabine,
mitoxantrone; CML-BC – blast crisis chronic myeloid leukemia; Dx – diagnosed; OS – overall
survival; DFS – disease free survival; RP2 – randomized phase 2.
Accepted Manuscript
Information Classification: General
Figure 1: CDK9 Pathway Schema
CDK9 forms a complex with Cyclin T1 known as P-TEFb, which is recruited and activated by both
Bromodomain-containing protein 4 (BRD4) and Mediator coactivator complex within a super-enhancer
complex of genes regulating transcription. P-TEFb phosphorylates RNA polymerase II (RNA Pol II)
which leads to its activation thereby promoting the transcription of genes regulating cell survival such as
c-MYC and MCL-1. MCL-1 inhibits the intrinsic pathway of apoptosis by neutralizing BAX/BAK and
thereby preventing mitochondrial outer membrane permeabilization (MOMP) and cytochrome c (Cyto C)
release. NOXA is a BH3 selective pro-apoptotic regulator of MCL-1. CDK9 inhibitors lead to the
inhibition of RNA Pol II-mediated transcription of c-MYC and MCL-1 ultimately promoting apoptosis
and preventing aberrant tumor-induced cell survival.
Figure 2: CDK6 Pathway Schema
The activity of CDK4/6 is regulated by Cyclin D proteins. Cyclin D1, D2, and D3 form a complex with
CDK4/6 leading to its activation. This complex of Cyclin D and CDK4/6 in turn phosphorylates
Retinoblastoma L86-8275 protein (RB), leading to its inactivation and freeing it from E2F, a transcription factor that
coordinates progression through the cell cycle from G1 to S phase. p16INK4A, a tumor suppressor gene,
inhibits CDK4/6, leading to dephosphorylated RB in complex with E2F, and thereby prevents cell cycle
progression to S phase. CDK4/6 inhibitors lead to cell cycle arrest at G1 phase by inhibiting