SUPPLEMENTARY APPENDIX

Table of Contents

Inclusion and Exclusion Criteria for the Study................................................................................3

Management of Tumor Lysis Syndrome and Diarrhea...................................................................5

Other Supportive Care.....................................................................................................................9

Management of Dose-Limiting Toxicities.......................................................................................9

Methods.........................................................................................................................................11

Minimal Residual Disease Analysis..........................................................................................11

Mitochondrial Priming...............................................................................................................11

Statistical Analysis.........................................................................................................................12

Supplemental Tables......................................................................................................................13

Supplemental Table S1.- Treatment-Emergent Grade ≥1 Non-Hematologic Toxicities Related

to the Study Drug Regimen........................................................................................................13

Supplemental Table S2 - Central Analysis of Minimal Residual Disease.................................16

Supplemental Table S3 - Outcomes of MRD-Negative CR Patients in Central MRD Cohort. 17

Supplemental Figures....................................................................................................................18

Supplemental Figure S1 - Heatmap of all patients with baseline AML mutations....................18

Supplemental Figure S2 - Heatmap of Overall Response and MCL-1 Dependence among

Patients based on the Genomic Classification by Papaemmanuil et al.32...................................19

Supplemental Figure S3 - MCL-1 priming in patients with and without CR............................20

Page 1 of 19

Inclusion and Exclusion Criteria for the Study

To be eligible for participation in the study, patient had to meet all of the following inclusion

criteria:

1. Were between the ages of ≥18 and ≤65 years

2. Had an established, pathologically confirmed diagnoses of AML by World Health

Organization (WHO) criteria with ≥20% bone marrow blasts based on histology or flow

cytometry

3. Were newly diagnosed and previously untreated

4. Had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤2

5. Had a serum creatinine level ≤1.8 mg/dL

6. Had an alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level ≤5

times upper limit of normal (ULN)

7. Had a total bilirubin level ≤2.0 mg/dL (unless secondary to Gilbert syndrome, hemolysis,

or leukemia)

8. Had a left ventricular ejection fraction (LVEF) >45% by echocardiogram (ECHO) or

multigated acquisition (MUGA) scan

9. Were nonfertile or agree to use an adequate method of contraception. Sexually active

patients and their partners must use an effective method of contraception associated with

a low failure rate prior to study entry, for the duration of study participation, and for at

least 6 months after the last dose of study drug.

10. Were able to comply with the requirements of the entire study.

Page 2 of 19

11. Provided written informed consent prior to any study related procedure. (In the event that

the patient is re-screened for study participation or a protocol amendment alters the care

of an ongoing patient, a new informed consent form must be signed.)

Exclusion Criteria:

Patients meeting any one of these exclusion criteria were prohibited from participating in the

study if they had:

1. Received any previous treatment for AML

2. Diagnosed with APL-M3 or CBF-AML

3. Required concomitant chemotherapy, radiation therapy, or immunotherapy. Hydroxyurea

is allowed up to the evening before starting (but not within 12 hours) of starting Induction

therapy.

4. Received >200 mg/m2 equivalents of daunorubicin

5. Had a peripheral blast count of >30,000/mm3 (may use hydroxyurea as in #3 above)

6. Had active central nervous system (CNS) leukemia

7. Had evidence of uncontrolled disseminated intravascular coagulation

8. Had an active, uncontrolled infection

9. Had other life-threatening illness

10. Had other active malignancies or diagnosed with other malignancies within the last 6

months, except nonmelanoma skin cancer or cervical intraepithelial neoplasia

11. Had mental deficits and/or psychiatric history that may compromise the ability to give

written informed consent or to comply with the study protocol.

12. Was pregnant and/or nursing.

Page 3 of 19

Management of Tumor Lysis Syndrome and Diarrhea

Tumor Lysis Syndrome:

Tumor lysis may occur as part of initial cytoreductive therapy. The most extreme form, known as

Tumor Lysis Syndrome (TLS), is characterized by hyperkalemia, hyperuricemia,

hyperphosphatemia, increased lactate dehydrogenase (LDH), coagulopathy, and a potential

cytokine release syndrome.

Mandatory IV hydration with 0.45% NaCl (or similar hydration fluid per institutional standard)

sterile solution at 100 cc/hour for at least 10 hours prior to initiation of the first dose of

chemotherapy during Induction (optional for subsequent cycles including Reinduction). If, by

Day 4, there is no evidence of tumor lysis syndrome, the hydration rate was reduced to a

maintenance level. Diligent monitoring of urine output frequently to ensure that fluid output

equals fluid input. If input was greater than output by 10%, administration of diuretics was

encouraged. Replacement of excessive fluid losses, including from diarrhea, was done unless

otherwise clinically indicated.

Mandatory allopurinol orally each day of dosing for first cycle was started at same time

as initiation of IV hydration

Mandatory oral phosphate binder was started at the same time as initiation of IV

hydration, unless contraindicated.

Evaluation of laboratory indicators of tumor lysis syndrome (TLS) during Induction

therapy. Evaluation of laboratory indicators of TLS was adjusted for Reinduction and

Consolidation therapy based on extent of tumor burden.

Page 4 of 19

o Tumor lysis laboratory evaluations (‘tumor lysis labs’) included electrolytes

(sodium, potassium, chloride, and carbon dioxide) as well as creatinine, calcium,

lactate dehydrogenase (LDH), uric acid, and phosphorus levels.

o For patients at high risk for TLS including those with risk scores of ≥2, those with

monocytic leukemia phenotype, or those with a history of FLT3 positive AML:

A STAT serum potassium was obtained at end of alvocidib infusion

Strongly recommend serum potassium at 2 hours post end of alvocidib

infusion as best practice

Full TLS panel was obtained at 4 hours post end of alvocidib infusion

If evidence of clinically meaningful TLS, obtaining TLS panel every 2

hours during the first 24 hours post end of alvocidib infusion (LDH levels

were recommended to be assessed at least once every 24 hours).

If no evidence of clinically meaningful TLS, a TLS panel was obtained

every 4 hours during the first 24 hours post end of alvocidib infusion

(LDH levels are recommended to be assessed at least once every 24

hours).

If, after the first 24 hours post end of alvocidib infusion, there was no

evidence of TLS, a TLS panel was obtained approximately every 6 hours

for the remainder of alvocidib treatment and then every 12 hours after

completion of alvocidib and during daunorubicin and cytarabine (LDH

levels are recommended to be assessed at least once every 24 hours).

Fibrinogen levels were monitored at baseline and then as clinically

indicated.

Page 5 of 19

Patients who were determined to be at intermediate- or high risk for TLS

were considered for rasburicase prophylaxis according to institutional

standards.

o For patients NOT at high risk for TLS:

During the first 24 hours - Tumor lysis labs were monitored at the start of

alvocidib and approximately every 4 hours. Laboratory studies during this

period were run as a “STAT” to ensure the results are available in a timely

manner.

If no evidence of TLS during first 24 hours, then tumor lysis labs were

monitored approximately every 6-8 hours for the remainder of alvocidib

treatment and then every 12 hours after the completion of alvocidib and

during cytarabine and daunorubicin.

Fibrinogen levels were monitored at baseline and then as clinically

indicated.

Risk of TLS and Guidelines for Management of High-Risk Patients

TLS management during treatment with alvocidib was implemented in previous studies, which

included medical prophylaxis for hyperuricemia, as well as aggressive monitoring and

management of hyperkalemia and other biochemical laboratory abnormalities. Rapid

development of hyperkalemia has been of particular concern in earlier studies. While these

guidelines are not necessarily consistent with specific standard recommendations for the

treatment of TLS, they are recommended based on previous experience with the treatment of

patients with alvocidib. These measures resulted in a lower incidence of TLS without adverse

outcomes.

Page 6 of 19

For this reason, investigators were encouraged to follow the recommended guidelines below but

could follow their own institution’s protocols in determining the best treatment for their patients.

If potassium levels were increased to >4.0 mEq/L, patients received a 30-gm dose of

sodium polystyrene sulfonate, unless there were other likely causes of hyperkalemia other

than TLS or contraindication to its use.

If potassium levels rose to >5.0 mEq/L, in addition to the 30-gm dose of sodium

polystyrene sulfonate, patients also received 10 units of IV rapid-acting insulin and

25 gm (one ampule) of IV dextrose 50%, unless there were other likely causes of

hyperkalemia other than TLS or contraindication to its use.

If potassium levels rose to >5.5 mEq/L, patients were considered for emergent

intermittent or continuous dialysis.

Calcium supplementation was only given for symptomatic hypocalcemia in this setting to

avoid renal precipitation of calcium phosphate crystals.

Patients who developed clinical evidence of cytokine release syndrome or who had

hyperkalemia requiring dialysis received immediate steroid therapy with an equivalent of

at least 20 mg of IV dexamethasone.

Diarrhea

If diarrhea occurred during therapy, patients had to initiate loperamide (or equivalent) 2 mg by

mouth every 2 hours during the waking hours. The rapid introduction of loperamide (or

equivalent) at the first signs of diarrhea was strongly encouraged. Once the diarrhea was

controlled, the time interval of loperamide was titrated to a frequency that adequately controlled

the diarrhea. The diarrhea observed with alvocidib almost always resolves following completion

of therapy, so treatment with loperamide following completion of therapy will not be required in

Page 7 of 19

most patients. If loperamide (or equivalent) did not control diarrhea, cholestyramine (or

equivalent) 5 gm orally four (4) times daily was added. For patients developing diarrhea during

alvocidib administration, subsequent treatments had to include a similar diarrhea prophylaxis. If

diarrhea was not controlled with the above prophylactic regimen and was grade 2 or greater,

therapy was to be held until diarrhea has resolved. Replacement of excessive fluid losses was

done unless otherwise clinically indicated.

Other Supportive Care

Prophylactic antibacterial and antiviral agents were administered to all patients prior to

chemotherapy. Anti-fungal prophylactics were administered per institutional/investigator

discretion. Growth factor support was allowed only after day 35 of therapy in the presence of

infection with ongoing neutropenia.

Management of Dose-Limiting Toxicities

Dose-limiting toxicities (DLTs) for alvocidib and 7+3 were determined during Induction and

Reinduction therapy, determined to be at least possibly related to study drug and were defined as

follows based on the NCI CTCAE version 4.03:

Any Grade 4 nonhematologic toxicity considered at least possibly drug related

Any Grade 3 nonhematologic toxicity considered at least possibly drug related and that

did not resolve to ≤Grade 2 within 48 hours, with the following exceptions:

o Grade 3 diarrhea, mucositis, nausea or vomiting was considered dose-limiting

only if resolution to <Grade 2 (including use of supportive care) required more

than 7 days

Page 8 of 19

Grade 4 cytopenias lasting ≥50 days from the start of the cycle in the absence of residual

leukemia if subject received one cycle of induction.

Grade 4 cytopenias lasting ≥60 days in the absence of residual leukemia if subject

received 2 cycles of induction

Grade 3 or 4 tumor lysis syndrome or related electrolyte disturbances (e.g., hyperkalemia,

hypophosphatemia, hyperuricemia) that did not resolve to ≤Grade 2 within 14 days

≥Grade 3 elevations in creatinine that did not resolve to ≤Grade 2 within 7 days

Any AST and ALT elevation ≥5 ×ULN accompanied by serum bilirubin levels

>2 ×ULN, regardless of duration

Any Grade 5 toxicity that was not clearly and incontrovertibly related to the underlying

disease or extraneous causes

Anorexia, alopecia, fever, neutropenic fever, and infections of any grade were not

designated as DLTs since they were known and expected toxicities associated with the

study drugs.

Page 9 of 19

Methods

Minimal Residual Disease Analysis

Samples were first incubated with antibodies for 20 minutes at room temperature. Red blood

cells were then lysed at 37°C using buffered NH4Cl for 5 minutes, followed by centrifugation at

300g. Cells were subsequently washed with PBS supplemented with 2% FCS and resuspended in

1% paraformaldehyde. Samples were analyzed on a FACSCaliburTM flow cytometer (Becton

Dickinson Biosciences). 200,000 events were analyzed for each sample. Data analysis was

performed with WinListTM software (Verity Software House). Two independent reviewers

viewed each sample to arrive at a consensus on the data.

Mitochondrial Priming

When added to cells, T-MS1 crosses the plasma membrane and antagonizes MCL-1, leading to

mitochondrial outer membrane pore (MOMP) formation and depolarization of the mitochondria.

Mitochondrial potential was assessed using the cationic dye, DiOC2(3) (ThermoFisher, Waltham,

MA, USA). Fresh AML patient’s BM cells were interrogated with T-MS1 treatment and the

measurement of the resulting change in mitochondrial potential was compared to carbonyl

cyanide m-chlorophenyl hydrazone (CCCP, Selleckchem, Houston, TX, USA) and water treated

controls. At least 2x106 viable cells were assessed per patient. Priming was calculated as:

% Priming=( ( Average % Polarized Water−% Polarized Peptide )Average % Polarized Water )×100

For concordance with prior priming methodology and values, an additional calibration factor of

1.6 was applied to patient priming values.

Page 10 of 19

Statistical Analysis

Maximum tolerated dose (MTD) for alvocidib was estimated by the highest alvocidib dose for

which the incidence of DLT during Cycle 1 was less than 33% in combination with 7+3

Page 11 of 19

Supplemental Tables

Supplemental Table S1.- Treatment-Emergent Grade ≥1 Non-Hematologic Toxicities Related to the Study Drug Regimen.

ToxicityaAlvocidib

<30/60 mg/m²(N=9)

Alvocidib30/60 mg/m²

(N=23)

StudyTotal

(N=32)Cardiac disorders

Palpitations 1 (11.1%) 0 (0.0%) 1 (3.1%)Eye disorders

Dry eye 0 (0.0%) 1 (4.3%) 1 (3.1)Eye pruritus 0 (0.0%) 1 (4.3%) 1 (3.1%)Keratitis 1 (11.1%) 0 (0.0%) 1 (3.1%)Lacrimation increased 1 (11.1%) 0 (0.0%) 1 (3.1%)Ocular hyperemia 1 (11.1%) 0 (0.0%) 1 (3.1%)Vision blurred 1 (11.1%) 0 (0.0%) 1 (3.1%)

Gastrointestinal disordersAbdominal pain 0 (0.0%) 6 (26.1%) 6 (18.8%)Ascites 0 (0.0%) 1 (4.3%) 1 (3.1%)Colitis/Enteritis/Enterocolitis 2 (22.2%) 4 (17.4%) 6 (18.8%) Constipation 3 (33.3%) 2 (8.7%) 5 (15.6%)Diarrhea 6 (66.7%) 23 (100%) 29 (90.6%)Dry mouth 0 (0.0%) 3 (13.0%) 3 (9.4%)Dyspepsia 0 (0.0%) 4 (17.4%) 4 (12.5%)Epigastric discomfort 0 (0.0%) 1 (4.3%) 1 (3.1%)Gastroesophageal reflux disease 0 (0.0%) 2 (8.7%) 2 (6.3%)Hematochezia 0 (0.0%) 1 (4.3%) 1 (3.1%)Lower gastrointestinal hemorrhage 1 (11.1%) 0 (0.0%) 1 (3.1%)Nausea 4 (44.4%) 16 (69.6%) 20 (62.5%)Proctalgia 1 (11.1%) 0 (0.0%) 1 (3.1%)Proctitis 1 (11.1%) 0 (0.0%) 1 (3.1%)Stomatitis 2 (22.2%) 4 (17.4%) 6 (18.8%)Vomiting 0 (0.0%) 13 (56.5%) 13 (40.6%)

General disorders and administration site conditions

Chills 1 (11.11%) 1 (4.3%) 2 (6.3%)Fatigue 4 (44.4%) 7 (30.4%) 11 (34.4%)Mucosal inflammation 0 (0.0%) 2 (8.7%) 2 (6.3%)Pyrexia 1 (11.1%) 2 (8.7%) 3 (9.4%)

Hepatobiliary disordersHyperbilirubinemia 2 (22.2%) 3(13.0%) 5 (15.6%)Alanine aminotransferase increased 1 (11.1%) 0 (0.0%) 1 (3.1%)Aspartate aminotransferase increased 1 (11.1%) 2 (8.7%) 3 (9.4%)Gamma-glutamyltransferase increased 0 (0.0%) 1 (4.3%) 1 (3.1%)

Page 12 of 19

ToxicityaAlvocidib

<30/60 mg/m²(N=9)

Alvocidib30/60 mg/m²

(N=23)

StudyTotal

(N=32)International normalized ratio increased 0 (0.0%) 1 (4.3%) 1 (3.1%)

Immune system disordersCytokine release syndrome 0 (0.0%) 1 (4.3%) 1 (3.1%)

Infections and infestationsBacteremia/Sepsis 4 (44.4%) 4 (17.4%) 8 (25.0%) Candida infection/Oral candidiasis 0 (0.0%) 2 (8.7%) 2 (6.3%) Cellulitis 0 (0.0%) 1 (4.3%) 1 (3.1%)Clostridium difficile infection 0 (0.0%) 1 (4.3%) 1 (3.1%)Conjunctivitis 0 (0.0%) 1 (4.3%) 1 (3.1%)Folliculitis 0 (0.0%) 1 (4.3%) 1 (3.1%)Fungemia 1 (11.1%) 0 (0.0%) 1 (3.1%)Lung infection and Pneumonia 0 (0.0%) 3 (13.0%) 3 (9.4%)

Metabolism and nutrition disordersDecreased appetite 1 (11.1%) 4 (17.4%) 5 (15.6%)Hyperkalemia 2 (22.2%) 4 (17.4%) 6 (18.8%)Hypermagnesemia 1 (11.1%) 0 (0.0%) 1 (3.1%)Hypoalbuminemia 2 (22.2%) 1 (4.3%) 3 (9.4%)Hypocalcemia 3 (33.3%) 3 (13.0%) 3 (9.4%)Hypokalemia 4 (44.4%) 3 (13.0%) 7 (21.9%)Hypomagnesaemia 2 (22.2%) 2 (8.7%) 4 (12.5%)Hyponatremia 1 (11.1%) 0 (0.0%) 1 (3.1%)Hypophosphatemia 3 (33.3%) 0 (0.0%) 3 (9.4%)Tumor lysis syndrome 1 (11.1%) 10 (43.5%) 11 (34.4%)Weight decreased 0 (0.0%) 2 (8.7%) 7 (21.9%)

Musculoskeletal and connective tissueArthralgia 1 (11.1%) 0 (0.0%) 1 (3.1%)

Nervous system disordersDisturbance in attention 1 (11.1%) 0 (0.0%) 1 (3.1%)Dysgeusia 2 (22.2%) 3 (13.0%) 5 (15.6%)Headache 1 (11.1%) 1 (4.3%) 2 (6.3%)Paresthesia 0 (0.0%) 1 (4.3%) 1 (3.1%)Syncope 1 (11.1%) 0 (0.0%) 1 (3.1%)

Renal and urinary disordersAcute kidney injury and blood creatinine increased

0 (0.0%)3 (13.0%) 3 (9.4%)

Hematuria 0 (0.0%) 1 (4.3%) 1 (3.1%)Respiratory, thoracic and mediastinal disorders

Dyspnea 1 (11.1%) 0 (0.0%) 1 (3.1%)Hiccups 0 (0.0%) 1 (4.3%) 1 (3.1%)Oropharyngeal pain 0 (0.0%) 1 (4.3%) 1 (3.1%)Pleural effusion 0 (0.0%) 1 (4.3%) 1 (3.1%)Pulmonary edema 0 (0.0%) 1 (4.3%) 1 (3.1%)

Page 13 of 19

ToxicityaAlvocidib

<30/60 mg/m²(N=9)

Alvocidib30/60 mg/m²

(N=23)

StudyTotal

(N=32)Sputum culture positive 1 (11.1%) 0 (0.0%) 1 (3.1%)

Skin and subcutaneous disordersAlopecia 1 (11.1%) 1 (4.3%) 2 (6.3%)Dermatitis 0 (0.0%) 1 (4.3%) 1 (3.1%)Erythema 0 (0.0%) 1 (4.3%) 1 (3.1%)Macule 0 (0.0%) 1 (4.3%) 1 (3.1%)Pruritus 1 (11.1%) 1 (4.3%) 2 (6.3%)Rash generalized 0 (0.0%) 2 (8.7%) 2 (6.3%)Rash maculo-papular 2 (22.2%) 2 (8.7%) 4 (12.5%)

Vascular disordersFlushing 1 (11.1%) 1 (4.3%) 2 (6.3%)

Other Epistaxis 0 (0.0%) 2 (8.7%) 2 (6.3%)

a Toxicity was classified using MedDRA = Medical Dictionary for Regulatory Activities (v19.1)

Page 14 of 19

Supplemental Table S2 - Central Analysis of Minimal Residual Disease

CR CharacteristicsCentral MRD Analysis Cohort

(N=9)

MRD-Negative CR 8/9 (89%)

Age

<60 years 6/7 (85.7%)

>60 years 2/2 (100.0%)

Secondary AML 0/0 (0.0%)

AML with MRC 1/1 (100.0%)

ELN risk

Favorable 4/4 (100.0%)

Intermediate 1/1 (100.0%)

Adverse 3/4 (75.0%)

SWOG cytogenetics risk

Favorable 0/0 (0.0%)

Intermediate 7/7 (100.0%)

Unfavorable 1/2 (50.0%)

Unknown 0/0 (0.0%)ELN=European LeukemiaNet, MRC= myelodysplasia-related changes, MRD= minimal residual disease, SWOG=Southwestern Oncology Group

Page 15 of 19

Supplemental Table S3 - Outcomes of MRD-Negative CR Patients in Central MRD Cohort

Outcomes Central MRD analysis cohort (n=9) *Allogenic stem cell transplantation 3 (33.3%)Received Consolidation Therapy 5 (55.6%)Relapse 0 (0.0%)

*Out of the 9 patients in the central MRD analysis cohort who achieved CR, one patient achieved CR with MRD while 8 achieved CR with no MRD. The patient who had MRD-positive CR received an allo-SCT and relapsed after allo-SCT.

Page 16 of 19

Supplemental Figures

Supplemental Figure S1 - Heatmap of all patients with baseline AML mutations

The frequency of baseline AML mutations determined for CR versus those without a CR. Colors

in the legend to the right represent the percentage of patients with a mutation achieving CR or

No CR. 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.

.

Page 17 of 19

Supplemental Figure S2 - Heatmap of Overall Response and MCL-1 Dependence among Patients based on the Genomic Classification by Papaemmanuil et al.32

Color legends to the right represent the genomic class of all the patients as per Papaemmanuil et

al 32. Patients achieving CR and those with MCL- 1 dependence are shown in green while

patients with no CR and MCL-1 non-dependent are shown in red.

Page 18 of 19

Supplemental Figure S3 - MCL-1 priming in patients with and without CR.

A box-and-whisker plot of the MCL-1 scores from response-evaluable patients with CR (n=18)

versus no CR (n=9). MCL-1 scores for 4 patients with CR were not available and are excluded.

Page 19 of 19