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Phase 1 Study of Alvocidib Followed by 7+3 (Cytarabine + Daunorubicin) in
Newly Diagnosed Acute Myeloid Leukemia
Joshua F. Zeidner1, Daniel J. Lee
2, Mark Frattini
2,3, Gil D. Fine
4, Judy Costas
4, Kathryn
Kolibaba4, Stephen P. Anthony
4, David Bearss
4, B. Douglas Smith
5
1 University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, NC
2Columbia University Medical Center, New York, NY
3Celgene, Summit, NJ
4Sumitomo Dainippon Pharma Oncology, Lehi, UT
5Johns Hopkins University School of Medicine, Sidney Kimmel Comprehensive Cancer Center,
Baltimore, MD
Running title: Alvocidib + 7+3 in Newly Diagnosed AML
Keywords: Alvocidib, AML, 7+3, CDK9, MCL-1
Authorship contributions:
J.F.Z. and D.L. contributed to study design, enrolled patients, collected and interpreted the data,
and wrote the manuscript. M.F. and B.D.S. contributed to study design, enrolled patients, and
wrote the manuscript. G.D.F. contributed to study design and interpreted the data. J.C. and K.K.
collected the data. S.A. and D.B. contributed to study design and wrote the manuscript. All
authors provided critical review and final approval of the manuscript.
Address for Correspondence:
Joshua F. Zeidner, MD
Associate Professor of Medicine
170 Manning Drive
Houpt Building, CB# 7305
Chapel Hill, NC 27599
Office: (919) 962-5164
Fax: (919) 966-6735
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Conflict of interest disclosures:
JFZ has received honoraria from AbbVie, Agios, Bristol Myers Squibb/Celgene, Daiichi-
Sankyo, Genentech, Pfizer, Takeda, and Tolero, consultancy fees from AsystBio Laboratories,
Celgene and Takeda, and institutional research funding from AROG, Forty Seven, Merck,
Takeda and Tolero.
DJL has served as a consultant for Celgene and received institutional research funding from
AbbVie, Bayer, Forty Seven, Genentech, Novartis, and Tolero.
BDS has received consulting fees from Jazz Pharmaceuticals, Novartis, and Celgene and
received research funding from Novartis, AbbVie, and Agios, and serves on a DSMB and
adjudication board for Celgene.
MF is an employee at Celgene.
GDF, JC, and DB are employees of Sumitomo Dainippon Pharma Oncology, formerly Tolero
Pharmaceuticals.
KK has received research funding from Acerta, Celgene, Genentech, Gilead, Novartis,
Pharmacyclics, Seattle Genetics, TG Therapeutics; consulting from Atara Biotech, Boston
Biomedical, TG Therapeutics, Tolero; and is employed by Compass Oncology
SPA is an employee of Sumitomo Dainippon Pharma Oncology, formerly Tolero
Pharmaceuticals, and consultant for Exact Sciences.
Word Count (Abstract): 237
Word Count (Main text): 3637
Total Number of Tables, Figures: 6
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Supplementary Files: 1
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TRANSLATIONAL RELEVANCE:
Standard frontline therapy for younger fit patients with newly diagnosed AML includes
induction chemotherapy with continuous infusion cytarabine and an anthracycline (i.e., 7+3)
with or without targeted agents such as midostaurin or gemtuzumab ozogamicin. However,
overall outcomes are poor with 5-year survival rates <50%. Alvocidib is a cyclin-dependent
kinase-9 (CDK9) inhibitor that leads to transcriptional suppression of MCL-1, an anti-apoptotic
BCL-2 family member that is up-regulated in AML. Prior studies showed that alvocidib followed
by cytarabine and mitoxantrone has anti-leukemic activity in both newly diagnosed and
relapsed/refractory AML. This phase 1 study revealed that alvocidib 30 mg/m2 IV over 30-
minutes followed by 60 mg/m2 IV over 4-hours on days 1-3 can be administered prior to 7+3
induction in newly diagnosed AML patients with overall CR rates of 69% in patients with non-
favorable cytogenetics. Preliminary clinical activity was seen in secondary AML patients who
have poor outcomes with conventional chemotherapy.
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ABSTRACT
Purpose: Alvocidib is a cyclin-dependent kinase-9 inhibitor leading to down-regulation of the
anti-apoptotic BCL-2 family member, MCL-1. Alvocidib has shown clinical activity in a timed-
sequential regimen with cytarabine and mitoxantrone in relapsed/refractory and newly diagnosed
AML but has not been studied in combination with traditional 7+3 induction therapy.
Experimental Design: A multi-institutional phase 1 dose-escalation study of alvocidib on days
1-3 followed by 7+3 (cytarabine 100 mg/m2/day IV infusion days 5-12 and
daunorubicin 60 mg/m2
IV days 5-7) was performed in newly diagnosed AML ≤65 years. Core-
binding factor AML was excluded.
Results: There was no maximum tolerated dose on this study; the recommended phase 2 dose of
alvocidib was 30 mg/m2 IV over 30-minutes followed by 60 mg/m
2 IV infusion over 4-hours.
There was 1 dose-limiting toxicity of cytokine release syndrome. The most common grade ≥3
non-hematologic toxicities were diarrhea (44%) and tumor lysis syndrome (34%). Overall,
69% (22/32) of patients achieved complete remission (CR). In an exploratory cohort, 8/9 (89%)
CR patients had no measurable residual disease, as determined by a centralized flow cytometric
assay. Clinical activity was seen in patients with secondary AML, AML with MDS-related
changes, and a genomic signature of secondary AML (50%, 50% and 92% CR rates,
respectively).
Conclusions: Alvocidib can be safely administered prior to 7+3 induction with encouraging
clinical activity. These findings warrant further investigation of alvocidib combinations in newly
diagnosed AML. This study was registered at clinicaltrials.gov identifier NCT03298984.
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INTRODUCTION
Overall outcomes remain poor in patients with Acute Myeloid Leukemia (AML) which affects
approximately 20,000 patients and more than 11,000 patients will die each year from this disease
in the United States (1). In younger patients (i.e., those <60-65 years), induction therapy with
7+3 remains the mainstay of treatment despite suboptimal long-term outcomes, particularly in
patients with adverse-risk disease whereby median overall survival (OS) is <1 year (2). In those
with a FLT3 mutation, midostaurin, an oral FLT3 inhibitor, improves OS but not complete
remission (CR) rates when added to 7+3 induction (3). However, FLT3 mutations are only
present in approximately 25-30% of newly diagnosed AML. The addition of Gemtuzumab
ozogamicin (GO), an antibody-drug-conjugate targeting CD33, to 7+3 induction was shown to
improve event-free survival (EFS) but not OS in newly diagnosed AML patients 50-70 years (4).
However, those with adverse-risk disease do not benefit from the addition of GO (5). Novel
agents are needed to improve clinical outcomes in the front-line treatment of AML, particularly
those with adverse-risk disease.
Alvocidib is a potent, non-selective cyclin-dependent kinase (CDK)-9 inhibitor with activity
against CDK4, 5, 7, 8 and 11. CDK9 forms a complex with cyclin T1 (PTEF-b) which is
recruited by bromodomain-containing protein-4 and mediator to superenhancer DNA complexes
to regulate the activity of RNA polymerase II. In turn, RNA polymerase II catalyzes the
transcription of genes regulating cell survival and proliferation, such as STAT3, c-MYC and
MCL-1. MCL-1 is up-regulated in AML and contributes to leukemia cell survival and resistance
to apoptosis (6,7) MCL-1 is also necessary for survival of leukemic stem cells, the population of
cells responsible for minimal residual disease (MRD), and facilitates AML progression (8).
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Blockade of MCL-1 has anti-leukemia activity in vivo (6,8). Inhibition of CDK9 prior to S-phase
specific cytotoxic chemotherapy agents may lead to synergistic apoptosis of leukemia cells (9).
Alvocidib has been investigated in a timed-sequential therapy (TST) approach in combination
with cytarabine and mitoxantrone (ACM) in 405 patients with both newly diagnosed (n=256)
and relapsed/refractory (n=149) AML with encouraging findings (10-17). To date, there have
been two dosing strategies done with alvocidib: a 60-minute IV bolus versus a “hybrid” dosing
of a 30-minute loading dose followed by a 4-hour infusion. Hybrid dosing was developed to
mitigate protein binding of alvocidib and maintain more sustained pharmacokinetic activity of
alvocidib (18). A randomized phase 2 trial of bolus versus hybrid ACM in newly diagnosed
poor-risk revealed similar clinical activity between both regimens though a lower dose of hybrid
alvocidib was studied compared with previous MTD seen and overall CR was non-significantly
higher in hybrid arm (74% vs. 62%, respectively) (15). Given the ease of administration, bolus
alvocidib was chosen for further development in newly diagnosed AML. Subsequently, a
randomized phase 2 clinical trial of bolus ACM led to higher CR rates compared with 1 or 2
cycles of 7+3 induction therapy (70% vs. 57%; p=0.07) in newly diagnosed AML patients with
non-favorable-risk cytogenetics (17,19). However, alvocidib has not been studied in the context
of conventional induction therapy with 7+3. Therefore, we designed a phase 1 dose-escalation
study of alvocidib followed by 7+3 in newly diagnosed AML patients with non-favorable-risk
cytogenetics to assess the safety, maximal tolerated dose (MTD), and clinical activity of this
regimen. We chose to utilize the hybrid dosing of alvocidib based on our prior experience.
MATERIALS AND METHODS
Study Population:
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This was a Phase 1, open-label, multicenter, dose-escalation study of alvocidib followed by
cytarabine/daunorubicin (7+3) in patients with AML (NCT03298984). Patients aged 18-65 years
with newly diagnosed, previously untreated AML were eligible. Hydroxyurea was permitted
prior to enrollment. Full eligibility criteria are listed in Supplementary Appendix. Patients were
excluded if they had previous treatment for AML, acute promyelocytic leukemia, or core-binding
factor AML. All patients provided written informed consent. This study was conducted as per the
Declaration of Helsinki after approval by ethics committee of each participating center.
Treatment Plan:
Alvocidib was dose-escalated starting at dose level 1: 20 mg/m2 30-minute IV bolus followed by
30 mg/m2 IV infusion over 4 hours on days 1-3 (Figure 1). Daunorubicin 60 mg/m
2 IV bolus
over 15 minutes was initiated on days 5-7, and cytarabine 100 mg/m2/day IV continuous 24-hour
infusion was administered on days 5-11. A BM aspirate/biopsy was performed on day 14 (+/-3
days) and patients with residual leukemia (>5% BM blasts and >10% cellularity) were
recommended to receive a second induction cycle with alvocidib days 1–3 (same dose level as
induction) followed by daunorubicin 45 mg/m2/day IV over 15 minutes on days 5-6 and
cytarabine 100 mg/m2/day IV continuous infusion days 5-9.
Patients who achieved CR received 2-4 cycles of consolidation therapy with high dose
cytarabine (HiDAC) 1.5-3 gm/m2 IV every 12 hours days 1, 3 and 5 upon full hematological
recovery. Allogeneic stem cell transplantation (alloSCT) was permitted after induction.
Toxicity and Response Assessments:
Dose limiting toxicities (DLT’s) were defined based on the NCI CTCAE version 4.03 and
outlined in Supplementary Appendix.
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Response assessment was performed by a BM aspirate/biopsy at the time of full hematologic
recovery or by day 50 and 60 of 1 versus 2 induction cycles, respectively, and assessed by
standardized ELN Guidelines (20).
Minimal Residual Disease (MRD) Analysis:
MRD was assessed in BM samples at the time of response by a uniform central assay
(Hematologics, Inc.) in an exploratory cohort, as has been previously described (21). 200,000
events were analyzed for each sample. The sensitivity of this assay was 0.02%. Details on the
methodology of this assay are included in the Supplementary Appendix.
Mitochondrial priming:
Leukemia dependence on BH3 member proteins was assessed, as previously described (22). For
evaluation of MCL-1 dependence, we utilized the MCL-1 binding protein, MS1, with
modifications allowing for improved cell penetrance, termed T-MS1 (23). T-MS1 has higher
potency and affinity for MCL-1 than NOXA (24). Details on methodology of this assay are
described in Supplementary Appendix.
Statistical Analysis:
The primary objective of this study was to establish an MTD of alvocidib prior to 7+3 induction.
Alvocidib dose was escalated using a 3+3 design (Figure 1). Successive cohorts of patients (3-6
per cohort) were treated with escalated doses until the MTD was established.
Secondary objectives included assessment of overall response rates (CR/CRi + partial remission
[PR]), OS, relapse-free survival (RFS) and EFS. Kaplan-Meier time-to-event analyses was
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performed on OS, EFS, RFS and duration of CR. Database lock was on April 30, 2020. All
statistical analyses were performed using SAS software version 9.4 or higher.
RESULTS
Patient Characteristics:
Between December 2017 and September 2019, 32 patients were enrolled to this study. Patient
characteristics are shown in Table 1. The median age was 58 years, 6 (19%) had secondary
AML, while 12 (38%) had AML with myelodysplasia-related changes (MRC) (defined by MDS-
related cytogenetics (25) or history of MDS/CMML). By ELN classification, 9 (28%) were
favorable, 7 (22%) were intermediate, and 16 (50%) were adverse-risk. Similarly, 12 (38%)
patients had unfavorable-risk cytogenetics by Southwest Oncology Group (SWOG) classification
(26). The most common mutations seen in this cohort were NPM1 (31%), ASXL1 (19%) and
RUNX1 (16%) mutations.
Safety:
The most common overall treatment-emergent adverse events included diarrhea (n=29, 91%,
Grade ≥3=14, 44%), nausea (n=20, 63%, Grade≥3=0, 0%), vomiting (n=13, 41%,
Grade≥3=0, 0%), fatigue (n=11, 34%, Grade≥3=0, 0%) and TLS
(n=11, 34%, Grade≥3=11, 34%) (Supplementary Table S1). The most common ≥grade 3
treatment-emergent adverse events were diarrhea (44%) and TLS (34%) (Table 2). These
toxicities resolved with supportive interventions as outlined in Methods and were not considered
dose-limiting. Diarrhea did not lead to any modifications or delays with alvocidib dosing. One
patient experienced a DLT (TLS, acute kidney injury, cytokine release syndrome: CRS) at the
highest alvocidib dose level studied (30 mg/m2 IV bolus followed by 60 mg/m
2 IV over 4 hours).
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Dexamethasone was initiated for CRS with rapid resolution of symptoms. The MTD was not
reached and the highest alvocidib dose level was determined to be the recommended Phase 2
dose (RP2D).
An expansion cohort enrolled 23 patients at the RP2D (Table 1). Overall, 30-day and 60-day
mortality was 3%. One patient died on day 26 due to septic shock during reinduction. In those
who achieved CR, the median time to partial and full neutrophil recovery (i.e., ≥0.5x109/L and
≥1x109/L, respectively) and partial and full platelet recovery (i.e., ≥50x10
9/L and ≥100x10
9/L,
respectively) was 34 and 36 days and 30 and 35 days.
Clinical Activity:
Among all enrolled patients, ORR and CR rates were 75% and 69%, respectively (Table 3). All
patients who achieved a CR achieved full hematologic recovery. Among response-evaluable
patients, the ORR and CR rates were 77% and 71%, respectively (one patient died on day 26 of
re-induction without a response assessment). Twenty-nine (91%) patients had no evidence of
residual leukemia on day 14 assessment (CR: 22/29 = 76% after 1 cycle of induction) whereas
3 (9%) received re-induction for residual leukemia on day 14. None of the patients who received
re-induction therapy achieved CR. Overall CR rates were 89% (8/9), 71% (5/7), and 56% (9/16)
for favorable-, intermediate-, and adverse-risk patients by ELN classification, respectively. Fifty
percent of patients with secondary AML (3/6) and AML with MRC (6/12) achieved CR. At the
RP2D, 15/23 (65%) achieved CR.
Of the 22 patients who achieved CR, 19 (86%) received consolidation therapy with intermediate
or HiDAC consolidation (median # of cycles: 2; Range: 1-4). Eleven (34%) proceeded to
alloSCT (ELN favorable-risk: n= 3, intermediate-risk: n=3, adverse-risk: n=5), all of whom
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achieved CR with induction therapy. Of the 11 patients who achieved CR and did not receive an
alloSCT, 5 (45%), 2 (18%), and 4 (36%) were ELN favorable-risk, intermediate-risk and
adverse-risk, respectively.
MRD Exploratory Cohort:
Twelve (38%) patients on the expansion cohort were included in a centralized MRD flow
cytometry assessment. Nine (75%) achieved CR and 8/9 (89%) were determined to be MRD-
negative (Intermediate-risk: 7/7; Adverse-risk: 1/2; Supplementary Table S3).
Genomic Signatures Predictive of Response:
A heatmap of genomic signatures obtained at diagnosis by institutional standard NGS panel
outlined by overall mutational landscape is demonstrated in Figure 2 and by proportion of CR
versus no CR in Supplementary Fig S1. As expected, the majority of patients with NPM1
mutations achieved CR (8/10 = 80% CR). Interestingly, overall CR rate was 83% (5/6) and
80% (4/5) among patients with ASXL1 and RUNX1 mutations, respectively. Only 1/3 (33%)
patients with a TP53 mutation achieved CR. Notably, among patients with a previously classified
genomic signature specific for secondary AML (i.e. ASXL1, BCOR, EZH2, SF3B1, SRSF2,
STAG2, U2AF1, or ZRSR2) (27), 11/12 (92%) achieved CR. Next, we analyzed overall response
among patients subdivided into the proposed genomic classification by Pappaemmanuil et al (28)
(Supplementary Fig S2). The most common genomically-defined subgroups in this cohort were
AML with NPM1 mutation (n=10) and AML with mutated chromatin, RNA-splicing genes, or
both (n=10). CR rates were 80% (8/10) and 90% (9/10) in patients classified as AML with
NPM1 mutation and AML with mutated chromatin, RNA-splicing genes, or both, respectively.
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Clinical Outcomes:
Figure 3 displays a Swimmer’s Plot of all patients enrolled (n=32). Of the 22 CR patients,
7 (32%) relapsed to date (median duration of CR: 8.6 months; Range: 1.4-13.6 months)]. Two
(18%) patients who achieved CR without MRD relapsed (including one who underwent an
alloSCT) while 9 (82%) who achieved CR without MRD remain in CR. Eleven (34%) patients
died. Causes of death were leukemia-related complications (n=9), septic shock during re-
induction therapy (n=1) and disseminated mucormycosis after consolidation therapy while in CR
(n=1).
Figure 3 depicts the OS, EFS and RFS of the 32 patients enrolled on this study. Mean and
median duration of follow-up was 11.4 and 9.2 months, respectively. The median OS was not
reached due to relatively short duration of follow-up. Landmark 1-year OS was
62.4% (95% CI: 41.9, 77.4%). Median EFS was 10.0 months (95% CI: 2.0, NA) while median
RFS was not reached. Landmark 1-year and 2-year EFS was 40.9 % (95% CI: 21.9, 59.1 %) and
34.1% (95% CI: 15.4, 53.8%) respectively. Landmark 1-year RFS was 59.5%
(95% CI: 31.7, 79.1%).
Mitochondrial Priming Correlates:
MCL-1 dependence was assessed by mitochondrial profiling from pre-treatment diagnostic BM
samples in 27/31 (87%) response-evaluable patients. Median MCL-1 score was 25.2% (range:
7.0-46.8%). There was no significant correlation of MCL-1 score among CR versus no CR
(Supplementary Fig S3). Twelve (44%) and 2 (7%) patients had MCL-1 priming scores >30%
and >40%, respectively. CR rate was 83% (10/12) and 67% (10/15) among those with MCL-1
dependence > and <30%, respectively. Eleven out of 12 patients with genomically-defined
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secondary AML (27) had MCL-1 analysis performed (median MCL-1: 31.5%; Range: 7-38.9%).
Of those analyzed, 7 (64%) had MCL-1 priming >30% while 4 (36%) were <30%.
DISCUSSION
Our findings demonstrate that alvocidib combined with standard 7+3 induction chemotherapy is
feasible and effective for younger patients (≤65 years) with newly diagnosed AML. As seen in
our previous studies with alvocidib in AML, the most frequent treatment-related AEs were
fatigue, nausea, diarrhea, and TLS (11-15,17,19). The most significant of these, TLS and
diarrhea, were manageable with appropriate prophylaxis and supportive interventions and were
not dose-limiting. TLS occurred rapidly after the first dose, was predominantly laboratory-based,
and generally resolved without clinical sequelae. Similarly, we observed a secretory diarrhea
associated with alvocidib, as seen previously (10), that occurs rapidly after the first dose and
responds to anti-diarrheal medications. There was 1 DLT of CRS leading to acute kidney injury
requiring temporary dialysis seen at the highest dose level. Although CRS was not separated
from adverse events documented as TLS in previous studies with alvocidib in AML, ≥grade 4
TLS was often accompanied by symptoms of CRS. In fact, infusional alvocidib has been shown
to increase pro-inflammatory cytokines, such as IL-6, potentially inciting an inflammatory milieu
that can lead to CRS (29). This is the first report to specify alvocidib-associated CRS in AML;
however, based on the toxicities seen in prior studies and noted TLS overlap, this is unlikely a
new phenomenon and rigorous monitoring for both TLS and CRS is required with alvocidib.
There was not an appreciable delay in hematologic recovery seen in patients on this study despite
the addition of alvocidib 3 days prior to 7+3 induction. The median time to partial neutrophil and
platelet recovery was 34 and 30 days, respectively, which is similar to CPX-351 (29) and TST
(17) and may be comparable to conventional 7+3 treatment regimens (30). Moreover, treatment-
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related mortality was low with only one (3%) patient expiring within 60 days of treatment. The
RP2D from this study – 30 mg/m2 IV bolus followed by 60 mg/m
2 IV over 4 hours – is
consistent with the MTD of a previous phase 1 study of alvocidib followed by cytarabine and
mitoxantrone (14).
The primary endpoint of this study was to establish an MTD and RP2D of alvocidib followed by
7+3 in newly diagnosed AML. Small numbers of patients enrolled on this study precluded
rigorous assessment of clinical activity of this regimen in comparison to historical controls.
Achieving CR is associated with longer RFS and OS when compared with non-responders and
those achieving CR without full recovery (31). All of the patients who achieved CR on this study
obtained full neutrophil and platelet recovery. The CR of alvocidib followed by 7+3 appears to
be similar to the composite CR of TST ACM treatment in newly diagnosed AML (69% vs. 68%)
though a direct comparison is not possible due to disparate patient populations studies (15,17).
Although we excluded patients with favorable-risk cytogenetics, it is worth noting that 28% of
enrolled patients were favorable-risk by ELN criteria due to either NPM1 mutation (n=8) or
CEBPA biallelic mutation (n=1). Notably, however, only 9% of patients on this study had
residual AML on day 14 BM assessment thus negating the need for re-induction therapy for the
vast majority of treated patients. In contrast, 25% and 44% of patients treated with ACM and
7+3, respectively, were found to have residual AML on day 14 in a randomized phase 2 study
(17). Re-induction therapy increases the risk of comorbidities such as organ toxicity, infectious
complications, and anthracycline-induced cardiotoxicity, delays hematologic recovery and
prolongs hospitalization. Increasing the efficacy of induction therapy without the need for
subsequent cytotoxic chemotherapy cycles would be highly advantageous.
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Patients with secondary AML and/or AML with MRC have dismal outcomes with conventional
chemotherapy. In a randomized phase 3 trial of cytarabine plus amonafide versus 7+3 in newly
diagnosed secondary AML, induction therapy with 7+3 yielded CR rates of 45% and median OS
of 7 months (32). Further, a randomized phase 3 study of CPX-351, liposomal cytarabine and
daunorubicin, versus 7+3 in newly diagnosed AML with MRC in patients 60-75 years revealed
CR rates of 37% versus 26%, respectively, and approximately 33% of patients in both arms
required re-induction therapy (33). In comparison, 50% of the patients with secondary AML or
AML with MRC achieved CR on this study though 50% and 42% were <60 years, respectively,
which could account for some of these differences.
Lindsley and colleagues previously reported that mutations in one of 8 genes (ASXL1, BCOR,
EZH2, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2) is highly specific for secondary AML (27).
Although ASXL1 mutations are defined as adverse-risk by ELN criteria, the other 7 mutations
specific for secondary AML are not specifically listed as adverse-risk by standardized criteria.
Recent data suggest that, among older patients with intermediate-risk AML, mutations associated
with secondary AML had significantly worse outcomes. A 2-class risk assessment from this
analysis defined patients with adverse-risk and those with intermediate-risk with secondary AML
mutations as “high-risk” disease (34). We noted an encouraging CR rate of 92% (11/12) in
patients with a genomic profile consistent with secondary AML. These findings are consistent
with previous studies showing particular clinical activity of alvocidib-containing induction
regimens in newly diagnosed secondary AML suggesting that the addition of alvocidib to
cytotoxic chemotherapy backbones may overcome at least some of the adverse-risk biology of
secondary AML (12,13,15,17).
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MRD determined by multicolor flow cytometry, quantitative PCR, or next-generation
sequencing is an important prognostic factor impacting the likelihood for relapse and survival
after induction and prior to alloSCT (35-38). Multicolor flow cytometric evidence of MRD in
CR1 leads to significantly worse outcomes compared with MRD-negative CR after induction
therapy (39-41). In a large prospective MRD analysis from the National Cancer Research
Institute (NCRI) AML17 trial, only 40% of younger patients treated with diverse “7+3”
induction therapy backbones achieved CR without MRD after 1 cycle. Further, 5-year OS was
63% versus 44% among patients with CR and MRD-negative versus MRD-positive after 1 cycle
of induction therapy (41). These studies reinforce the significant clinical impact of achieving an
MRD-negative CR after induction therapy and suggest that new approaches are needed to target
MRD. To evaluate alvocidib’s role in eliminating MRD, an exploratory cohort of 12 patients
were treated at the RP2D to prospectively assess MRD status after one cycle of induction by
centralized flow cytometry. Of these 12 patients, 9 (75%) achieved CR, and 8 (67%) achieved an
MRD-negative CR with a detection threshold of <0.02%. New approaches are imperative to
target leukemic cells in order to convert MRD-positive patients to an MRD-negative particularly
before alloSCT. Further study is warranted to specifically address whether the addition of
alvocidib to 7+3 can decrease MRD-positivity, lead to deeper responses and subsequently
improve clinical outcomes.
Based on alvocidib’s mechanism of action as a CDK9 inhibitor, we hypothesized that AML
patients whose leukemia cells are dependent on MCL-1 for survival may have a predilection for
response to alvocidib. Mitochondrial profiling assesses the relative dependence of anti-apoptotic
BCL-2 peptides in mediating cell survival within a tumor (43). NOXA is a BH3 sensitizer that
selectively binds to and antagonizes MCL-1 leading to apoptosis in cells dependent on MCL-1
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Page 18 of 30
for survival. A NOXA mimetic peptide (T-MS1) “primes” cells for apoptosis, and high priming
scores reflect cells that are considered to be MCL-1 dependent. We previously found that MCL-1
dependence was associated with response to treatment with TST ACM induction in newly
diagnosed AML (44). Thus, we performed an exploratory prospective analysis of MCL-1
dependence on response to alvocidib followed by 7+3. We defined the criteria of MCL-1
dependence as a priming threshold of >30%. Although there are no uniform criteria for defining
MCL-1 dependence, the 30% threshold is consistent with our revised eligibility criteria for a
randomized phase 2 trial of cytarabine plus mitoxantrone with or without alvocidib in patients
with relapsed/refractory MCL-1 dependent AML (45). We did not appreciate any significant
differences in response to alvocidib in patients with or without MCL-1 dependence though this
exploratory analysis was limited by small numbers of patients in each cohort. Further, we did not
assess for dependence on other pro-survival mechanisms such as BCL-2 or BCL-XL dependence
which should be further explored in future studies. Nonetheless, 83% (10/12) of patients with
MCL-1 dependence achieved CR. Given alvocidib’s multi-CDK inhibitory activity and
subsequent inhibition of RNA polymerase II, it is likely that alvocidib exerts anti-leukemia
activity more broadly than direct MCL-1 inhibition (9,46) and may provide a therapeutic
advantage over selective CDK9 and MCL-1 inhibitors even in patients considered to be MCL-1
dependent. A randomized phase 2 clinical trial is warranted comparing standard induction
chemotherapy with or without the addition of alvocidib with prospective evaluation and
stratification based on MCL-1 dependence.
In conclusion, alvocidib administration prior to 7+3 induction is tolerable, feasible, and showed
encouraging clinical activity in newly diagnosed AML with non-favorable risk cytogenetics.
TLS and diarrhea are the most common severe toxicities that can be managed adequately with
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supportive care measures. Most notably, alvocidib followed by 7+3 led to a 92% CR rate in a
genomically-defined signature of secondary AML that has traditionally poor clinical outcomes.
In an exploratory cohort, high rates of MRD-negative CR rates were obtained with alvocidib
followed by 7+3. These data warrant a randomized clinical trial of alvocidib with or without 7+3
in newly diagnosed AML.
ACKNOWLEDGEMENTS
This study was supported by Tolero Pharmaceuticals, acquired by Sumitomo Dainippon Pharma.
This data was presented in part at the annual 2019 European Hematology Association (EHA)
meeting in Amsterdam, Netherlands as a poster presentation and at the 2020 Virtual EHA
meeting as a poster presentation.
The authors would like to thank Dr. Judith Karp for her contribution to the study design and
development plan of alvocidib. We would like to thank the research staff and all co-investigators
at University of North Carolina, Johns Hopkins, and Columbia University. We would also like to
thank all patients and their families for trusting us in their care and allowing us to conduct this
study.
Sonali Lokhande MD, a medical writer from Criterion Edge supported by funding from
Sumitomo Dainippon Pharma Oncology/Tolero Pharmaceuticals, provided editorial assistance to
the authors during preparation of this manuscript.
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TABLES
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Table 1 Patient Characteristics
Patient Characteristics
Alvocidib Dose
<MTD
(<30/60mg/m2)
N=9
Alvocidib Dose at
MTD
(30/60mg/m2)
N=23
Study Total
N=32
Age, median (range), years 60 (31, 65) 51 (33, 65) 58 (31, 65)
Age >60, years 5 (55.6%) 6 (26.1%) 11 (34.4%)
Male, n (%) 4 (44.4%) 14 (60.9%) 18 (56.3%)
ECOG performance status
0 5 (55.6%) 8 (34.8%) 13 (40.6%)
1 3 (33.3%) 13 (56.5%) 16 (50.0%)
2 1 (11.1%) 2 (8.7%) 3 (9.4%)
Bone Marrow Blasts (%) median (range) 49 (23, 92) 47 (12, 98) 48 (12, 98)
Baseline WBC (x109/L)- median (range) 3.1 (1.60, 15.54) 3.99 (0.50, 16.00) 3.87 (0.50, 16.00)
Secondary AML, n (%) 2 (22.2%) 4 (17.4%) 6 (18.8%)
t-AML 0 (0.0%) 3 (13.0%) 3 (9.4%)
Prior MDS, n (%) 1 (11.1%) 2 (8.7%) 3 (9.4%)
Prior CMML 0 (0.0%) 1 (4.3%) 1 (3.1%)
Prior MPN 1 (11.1%) 0 (0.0%) 1 (3.1%)
AML with MRC, n (%) 4 (44.4%) 8 (34.8%) 12 (38.0 %)
Genomically-defined secondary AML 2 (22.2%) 10 (43.5%) 12 (38.0%)
ELN classification, n (%)
Favorable 3 (33.3%) 6 (26.1%) 9 (28.1%)
Intermediate 3 (33.3%) 4 (17.4%) 7 (21.9%)
Adverse-risk 3 (33.3%) 13 (56.5%) 16 (50.0%)
SWOG cytogenetics classification, n (%)
Favorable 0 (0.0%) 0 (0.0%) 0 (0.0%)
Intermediate 4 (44.4%) 16 (69.6%) 20 (63%)
Unfavorable 5 (55.6%) 7 (30.4%) 12 (38%)
Genetic mutations, n (%)
ASXL1 0 (0.0%) 6 (26.1%) 6 (18.8%)
CEBPA 0 (0.0%) 2 (8.7%) 2 (6.3%)
DNMT3A 2 (22.2%) 3 (13.0%) 5 (15.6%)
EZH2 0 (0.0%) 2 (8.7%) 2 (6.3%)
FLT3-ITD 0 (0.0%) 3 (13.0%) 3 (9.4%)
FLT3-TKD 1 (11.1%) 0 (0.0%) 1 (3.1%)
IDH1 1 (11.1%) 2 (8.7%) 3 (9.4%)
IDH2 0 (0.0%) 4 (17.4%) 4 (12.5%)
NPM1 3 (33.3%) 7 (30.4%) 10 (31.3%)
RUNX1 0 (0.0%) 5 (21.7%) 5 (15.6%)
TET2 0 (0.0%) 4 (17.4%) 4 (12.5%)
TP53 3 (33.3%) 1 (4.3%) 4 (12.5%)
U2AF1 1 (11.1%) 2 (8.7%) 3 (9.4%)
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Abbreviations: CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MPN, myeloproliferative
neoplasms; MRC, myelodysplasia-related changes; MTD, maximum tolerated dose.
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Table 2 Treatment-Emergent Grade >3 Non-Hematologic Toxicities
Toxicity, n (%)
Alvocidib Dose
<MTD
(<30/60mg/m2)
N=9
Alvocidib Dose at
MTD
(30/60mg/m2)
N=23
Study Total
N=32
Gastrointestinal disorders
Colitis and Enterocolitis 1 (11.1%) 1 (4.3%)0 2 (6.3%)
Diarrhea 3 (33.3%) 11 (47.8%) 14 (43.8%)
Stomatitis 1 (11.1%) 0 (0.0%) 1 (3.1%)
Infections and infestations
Bacteremia/Sepsis 3 (33.3%) 4 (17.4%) 7 (21.9%)
Cellulitis 0 (0.0%) 1 (4.3%) 1 (3.1%)
Clostridium difficile infection 0 (0.0%) 1 (4.3%) 1 (3.1%)
Fungaemia 1 (11.1%) 0 1 (3.1%)
Lung infection and Pneumonia 0 (0.0%) 3 (13.0%) 3 (9.4%)
Renal and urinary disorders
Acute kidney injury and increased blood
creatinine 0 (0.0%) 2 (8.7%) 2 (6.3%)
Hepatobiliary disorders
Aspartate aminotransferase increased 0 (0.0%) 2 (8.7%) 2 (6.3%)
Blood bilirubin increased 0 (0.0%) 1 (4.3%) 1 (3.1%)
Gamma-glutamyltransferase increased 0 (0.0%) 1 (4.3%) 1 (3.1%)
Tumor Lysis Syndrome 1 (11.1%) 10 (43.5%) 11 (34.4%)
Other
Epistaxis 0 (0.0%) 1 (4.3%) 1 (3.1%)
Immune system disorders
Cytokine release syndrome 0 (0.0%) 1 (4.3%) 1 (3.1%)
Metabolism and nutrition disorders
Hyperkalemia 0 (0.0%) 1 (4.3%) 1 (3.1%)
Hypoalbuminemia 0 (0.0%) 1 (4.3%) 1 (3.1%)
Hypocalcemia 2 (22.2%) 3 (13.0%) 5 (15.6%)
Hypokalemia 1 (11.1%) 1 (4.3%) 2 (6.3%)
Hypophosphatemia 2 (22.2%) 0 (0.0%) 2 (6.3%)
Nervous system disorders
Syncope 1 (11.1%) 0 (0.0%) 1 (3.1%)
Skin and subcutaneous tissue
disorders
Rash maculo-papular 0 (0.0%) 1 (4.3%) 1 (3.1%)
Abbreviations: MTD, Maximum tolerated dose. a Toxicity was classified using MedDRA = Medical Dictionary for Regulatory Activities (v19.1)
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Page 29 of 30
Table 3 Clinical Activity – Response Assessments
Response Characteristics Study Total
(N=32)
CR, n (%) 22 (68.8%)
CRi (%) 0 (0.0%)
Overall CR/CRi, n (%) 22 (68.8%)
PR, n (%) 2 (6.3%)
Overall Response Rate (CR+CRi+PR) 24 (75.0%)
Overall CR subgroups, n (%)
Evaluable Study Population, (n=31) 22 (71.0%)
At MTD, (n=23) 15 (65.2%)
Age <60 years, (n=21) 16 (76.2%)
Age >60 years, (n=11) 6 (54.5%)
Secondary AML, (n=6) 3 (50.00%)
AML with MRC, (n=12) 6 (50.0%)
Genomically-defined secondary AML 11 (92%)
ELN-risk, n (%)
Favorable (n=9) 8 (88.9%)
Intermediate (n=7) 5 (71.4%)
Adverse (n=16) 9 (56.3%)
SWOG cytogenetics risk, n (%)
Favorable (n=0) 0 (0.0%)
Intermediate (n=20) 16 (80.0%)
Unfavorable (n=12) 6 (50.0%)
Unknown (n=0) 0 (0.0%)
Abbreviations: CR, complete remission, MRD positive or unknown; CRi, complete remission with incomplete recovery;
ELN, European LeukemiaNet; MRC, myelodysplasia-related changes; MTD, maximum tolerated dose; PR, partial remission;
SWOG, Southwestern Oncology Group.
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Page 30 of 30
FIGURES:
Figure 1 Consort Diagram
Figure 2 Heatmap of Genomic Signatures and Response
A heatmap representing patients with baseline AML mutation achieving CR (green) or No CR
(red). Positive and negative AML mutations are shown in white and grey respectively.
Unknown/not tested mutations are shown in black. One patient who achieved a CR without
detectable MRD was not assessed for the full panel of 15 genes and was excluded from this
analysis.
Figure 3 Clinical Outcomes
Swimmer’s plot of best treatment response and survival for all 32 patients (A). The swim lanes
represent subjects in the study and indicate their progression and survival in the trial along with
response to therapy. The horizontal axis depicts survival in months since the first dose of
alvocidib. Colors of the swim lanes depict best response to treatment (orange – CR (MRD-
negative), blue – CR/CRi, green – PR, grey – no response). Solid black circles (●) on the swim
lanes represent earliest CR/CRi or PR, black triangles (▲) represent allogenic stem cell
transplantation, black circles (○) represent relapse/progression and red stars represent death.
Follow-up data (up to a protocol-specified maximum of 2 years) is still being accrued. Overall
survival (B), event-free survival (C) from Day 1 of treatment up to death, relapse, or no response
to treatment and relapse-free survival (D) defined as time from CR/CRi up to disease relapse or
death, for all patients using Kaplan-Meir estimates. Median OS, and RFS could not be
calculated.
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