Genomic Guided Therapy in Myeloma

• Dr. A. Keith Stewart

• Vasek and Anna Maria Professor of Cancer Research

• Carlson-Nelson Endowed Director, Center for Individualized Medicine

• Mayo Clinic

Improving response rates and CR with IMiD and Proteasome inhibitor combination therapies

1960-651965-701970-751975-801980-851985-901990-951995-002000-052005-10

Improving Survival in MM

25% of patients live less than 3 years

mSMART 2.0 Variable Outcomes in MM

• FISH• Del 17p• t(14;16)• t(14;20)

• GEP • High risk

signature

• Others• Hyperdiploid• t(11;14)• t(6;14)

• FISH• t(4;14)

• Cytogenetic deletion 13 or hypodiploidy

• PCLI ≥3%

High 20%

Intermediate20%

Standard60%

3 years 4-5 years 10 years

Todays Goal: To Describe Three Clinically Relevant, Multiple Myeloma Drug Resistance Mechanisms and Their Impact on Treatment Decision Making

•Clonal Tides

•Cereblon and IMiDs

•Proteasome Inhibitor Resistant MM Progenitor Cells

•Genomic Monitoring

Clinical Course of t(4;14) High Risk Patient

• 5 unique clones at diagnosis• Variable chemotherapy response• Minor drug resistant clone lethal

Clonal Tides

Implications

• Multiple clones with variable drug sensitivity

• Combination chemotherapy a necessity

• Resuscitation of drug sensitive clones

• Once resistant not always resistant

• Continuous suppressive therapy logical

• Minor drug resistant clones lethal

• Need to understand mechanism of resistance as a means to eradicate

9

Carfilzomib, Lenalidomide, and Dexamethasone vs Lenalidomide and

Dexamethasone in Patients withRelapsed Multiple Myeloma:

Interim Results from ASPIRE, a Randomized, Open-Label, Multicenter Phase

3 Study

A. Keith Stewart, S. Vincent Rajkumar, Meletios A. Dimopoulos, Tamás Masszi, Ivan Spicka, Albert Oriol, Roman Hájek, Laura Rosiñol, David S. Siegel, Georgi G. Mihaylov, Vesselina Goranova-Marinova, Péter Rajnics, Aleksandr Suvorov, Ruben Niesvizky, Andrzej Jakubowiak, Jesus F. San Miguel, Heinz Ludwig, Naseem Zojwalla, Margaret E. Tonda, Biao Xing,

Philippe Moreau and Antonio Palumbo

NEJM, Jan 8th, 2015

ASPIRE Study Design

10

RdLenalidomide 25 mg Days 1–21

Dexamethasone 40 mg Days 1, 8, 15, 22

KRdCarfilzomib 27 mg/m2 IV (10 min)

Days 1, 2, 8, 9, 15, 16 (20 mg/m2 days 1, 2, cycle 1 only)

Lenalidomide 25 mg Days 1–21

Dexamethasone 40 mg Days 1, 8, 15, 22

Randomization

N=792

Stratification:

• β2-microglobulin

• Prior bortezomib

• Prior lenalidomide

After cycle 12, carfilzomib given on days 1, 2, 15, 16

After cycle 18, carfilzomib discontinued

28-day cycles

Secondary Endpoints: Response

≥CR ≥VGPR ORR (≥PR)0

10

20

30

40

50

60

70

80

90

100

31.8

69.9

87.1

9.3

40.4

66.7

KRdRd

Per

cent

age

of P

atie

nts

11

P<.0001

P<.0001

sCR 14.1% vs 4.3%

P<.0001

Median duration of response was 28.6 months in the KRd group and 21.2 months in

the Rd group

Primary Endpoint: Progression-Free SurvivalITT Population (N=792)

12

1.0

0.8

0.6

0.4

0.2

0.0

Pro

port

ion

Sur

vivi

ngW

ithou

t P

rogr

essi

on

KRdRd

0 6 12 18 24 30 36 42 48Months Since Randomization

KRd Rd(n=396) (n=396)

Median PFS, mo 26.3 17.6HR (KRd/Rd) (95% CI) 0.69 (0.57–0.83)P value (one-sided) <0.0001

No. at Risk:KRd

Rd396 332 279 222 179 112 24 1396 287 206 151 117 72 18 1

Primary Endpoint: Progression-Free SurvivalITT Population (N=792)

13

1.0

0.8

0.6

0.4

0.2

0.0

Pro

port

ion

Sur

vivi

ngW

ithou

t P

rogr

essi

on

KRdRd

0 6 12 18 24 30 36 42 48Months Since Randomization

KRd Rd(n=396) (n=396)

Median PFS, mo 26.3 17.6HR (KRd/Rd) (95% CI) 0.69 (0.57–0.83)P value (one-sided) <0.0001

No. at Risk:KRd

Rd396 332 279 222 179 112 24 1396 287 206 151 117 72 18 1

PFS by Risk Group

14

KRd(n=396)

Rd(n=396)

Risk Group by FISH

NMedian, months

NMedian, months

HRP-value

(one-sided)

High 48 23.1 52 13.9 0.70 0.083

PFS by Risk Group

15

KRd(n=396)

Rd(n=396)

Risk Group by FISH

NMedian, months

NMedian, months

HRP-value

(one-sided)

High 48 23.1 52 13.9 0.70 0.083

Standard 147 29.6 170 19.5 0.66 0.004

Secondary Endpoints: Interim Overall Survival AnalysisMedian Follow-Up 32 Months

16

Median OS was not reached; results did not cross the prespecified stopping

boundary (P=0.005) at the interim analysis

1.0

0.8

0.6

0.4

0.2

0.0

Pro

port

ion

Sur

vivi

ng

KRdRd

0 6 12 18 24 30 36 42 48

Months Since Randomization

KRd Rd(n=396) (n=396)

Median OS, mo NE NEHR (KRd/Rd) (95% CI) 0.79 (0.63–0.99)P value (one-sided) 0.018

No. at Risk:KRd

Rd396 369 343 315 280 191 52 2396 356 313 281 237 144 39 3

Health-Related Quality-of-Life

17

EORTC Global Health Status improved in the KRd group vs the Rd group over 18 cycles of treatment (P=0.0001)

70

65

60

55

50

EORT

C Q

LQ-C

30 G

loba

l Hea

lth S

tatu

s/Q

ualit

y-of

-Life

Sco

re

Cycle 1(Baseline) Assessment Time Point (Day 1)

Carfilzomib group

Control group

Cycle 3 Cycle 6 Cycle 12 Cycle 18

Conclusions

18

PFS was significantly improved by 8.7 months with KRd

(HR, 0.69; P<0.0001)

‒An unprecedented median PFS of 26.3 months with

KRd

Interim OS analysis: trend in OS favoring the KRd

group; Kaplan-Meier 24-month OS rates 73.3% (KRd)

versus 65.0% (Rd)

ORR was higher with KRd (87.1% vs 66.7%);

significantly more patients achieved ≥CR (31.8% vs

9.3%)

QoL Global Health Status improved

Inferred Conclusions

19

Combination of drugs is better

Longer duration of combination therapy may

have helped

High Risk disease is still worse

Resistance eventually emerges

IMiD Structures

PotencySide effects Potency

Cereblon

• Cereblon on chromosome 3 was first described as associated with human intelligence (Cerebral protein with Lon protease)

• Functions in the brain as an ionic channel regulator

• Highly conserved from plants to humans, broadly expressed

• Forms an E3ligase complex with DDB1, Cul4A, Roc1

Cereblon Levels are Highest in MM, Leukemias, and Neuroblastoma

0

0.2

0.4

0.6

0.8

1

1.2

Via

bilit

y (%

con

trol

)

Lenalidomide

MM1.S Res

MM1.S

MM1.S MM1.S res

CRBN

b-actin

Zhu YX, et al. Blood. 2011;118:4771-9.

Lenalidomide Resistant MM Cells Lack Cereblon

<0.81 <0.9 >0.910 15 36

0

5

10

15

20

25

30

35

N =

CRBN expression as a percentage of the mean levels in all

MM

0%

19%

33%

Gene Expression Levels of Cereblon Predict Response Rate to Pomalidomide

Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28

0 12 24 36 48 600

20

40

60

80

100

Lowest 25%Top 25%

Months

% A

live

Gene Expression Levels of Cereblon Predict Overall Survival of Pomalidomide Treated Patients

0 12 24 36 48 600

20

40

60

80

100

Lowest 25%

Months

% A

live

P=0.01 P=0.005

9.1 vs 27 months

Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

Top Lenalidomide Regulated Putative CRBN Interacting Proteins

Proteins Experiment 1* Experiment 2* Experiment 3*

Regulation -Len +Len -Len +Len -Len +Len

GNL2 2 0 2 0 2 0 Down

STUB1 2 0 2 0 2 0 Down

IKZF1 5 0 6 0 3 0 Down

IKZF3 5 0 7 0 10 0 Down

ANKRD12 2 1 4 0 6 0 Down

KPNA2 15 2 16 9 36 19 Down

CUL4A 30 66 34 45 31 58 Up

CUL4B 18 43 9 45 44 130 Up

DDB1 112 179 82 261 122 271 Up

SQSTM1 11 17 7 17 12 21 Up

*The number in each column represents the number of assigned spectra for that protein

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

IKZF1/IRF4/Myc Degradation After Lenalidomide

IKZF3

IKZF1

IRF4

MYC

b-actin

Len - + - + - + - +

Time 3h 6h 24h 48h

CRBN/IZKF1/IRF4 Expression and Drug Resistance

IZKF1 L208R damaging mutation

XG1 MM1.S KMS11 H929 JJN3 EJM OCI-MY5 FR4 SKMM

IZKF1

IZKF3

b-actin

Len Sensitive Len Resistant

CRBN

IRF4

t(6;14) IgH-IRF4 translocation

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

Degradation of Ikaros by Cereblon Binding Small Molecules

1 2 3 4 5 6 7 8 9 10 110

200

400

600

800

1000

1200

1400

1600

1800

ThalLenPom

8226

KMS12PE

EJM My5

SKMM

2JJN

3FR

4H112

OPM2

U266

KMS18

H929

MM

1.S

MM

1.R

KMS11

XG10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Len 2uM 5hrs

Sensitivity of MM to Lenalidomide is Correlated with IKAROS Degradation Efficiency

Most resistant cell lines Most Sensitive cell lines

• MM cell lines with adenoviral vector expressing IKAROS 1 - luciferase fusion gene. Luciferase activity was measured and normalized to cells treated with DMSO

Proteasome Inhibitors block IKAROS degradation by Lenalidomide

0 1 2 3 4 5 6 7 80

200

400

600

800

1000

1200

1400

1600

1800

0

Len (2uM)

Bortezomib Concentration (nM)

0 2.5 5 10 15 20 25 30 35 400

200

400

600

800

1000

1200

1400

1600

1800

2000

CarfilzomibCar+Len 2uM

Carfilzomib concentration: nM

• Cereblon is the IMiD target and accumulates with IMiD binding

• Ikaros is rapidly degraded by CRBN in presence of IMiD – a process blocked by bortezomib or carfilzomib

• Low Cereblon and Ikaros levels may predict response and survival

• Resistance can be explained in many cases by disruption of this pathway

Summary

What About Proteasome Inhibitor Resistance ?

Druggable genome siRNA screening

Day 0siRNA - preprinted+ Transfection reagent+ Cells / fresh media

Day 4Assess viabilityCellTitre Glo

IncubateIn bortezomib

Human Druggable Genome siRNA Set

Bortezomib

Synthetic Lethal siRNA Screens Identify IRE1 as Essential for Bortezomib Cytotoxicity in MM Cells

Kinome(650 genes)

IRE

1B

orte

zom

ib

← Sensitizing kinases (on siRNA knockdown)

Resistance kinases (on siRNA knockdown) →

Bor

tezo

mib IR

E1

Druggable Genome(7000 genes)← Sensitizing genes

(on knockdown)Resistance genes

(on SiRNA knockdown) →

XBP1u XBP1s Plasma Cells

IRE1Unfolded Protein Response

Tiedeman et al: Cancer Cell (2013) Sep 9; 24, 289-304

• Loss of IRE1/XBP1s confers PI resistance

• Low IRE1/XBP1s cellsare pre plasmablasts

• These XBPs low MM progenitors survive PI and contribute to relapse

• High immunoglobulinproduction confers sensitivity

Proteasome Inhibitor Resistance

Tiedeman et al: Cancer Cell (2013) Sep 9; 24, 289-304

Cancer Cell (2013) Sep 9; 24, 289-304

MM Progenitors and Role in Therapeutic Resistance

Scottsdale, Arizona Rochester, Minnesota Jacksonville, Florida

Mayo Clinic College of MedicineMayo Clinic Comprehensive Cancer Center

KM Kortüm, AK Stewart, LA Bruins, G Ahmann, G Vasmatzis, SV Rajkumar, S Kumar, A Dispenzieri, MQ Lacy, MA Gertz, R Fonseca, M Champion, PL Bergsagel and E Braggio;

A Custom Multiple Myeloma Mutation Panel for Clinical Targeted Sequencing

Publication of 300+ MM genomes/exomes essentially defined the genetic landscape of

the disease

Only a limited set of genes is recurrently mutated in MM

Lohr et al., Cancer Cell 2014

Myeloma Mutation Panel (M3P)

• Recurrently mutated : FAM46C, DIS3, TP53, RB1, etc.

• Actionable: BRAF, RAS, IDH1/2, FGFR3, AKT etc.

• Pathways: NF-kB, MAPK, IL6-JAK-STAT, MYC, etc.

• Copy Number: Hyperdiploid, del17, del13, 1q+

• Drug Resistance: IMiDs, Proteasome inhibitors,

Glucocorticoid

77 genes, 1271 amplicons, 271 Kb

Customized MM specific gene panel using semiconductor sequencing technology

• Mutation and copy-number information

• Low input DNA quantity (~10 ng)

• Results available in clinically meaningful timeframes: 3 days from library preparation to data analysis

• Low cost (~$250/sample)

• Increased coverage

Results To Date:

• 142 untreated and 14 double-refractory MM patients

incl. corresponding germline sample

• Average sequencing depth : 600X

• 283 nonsynonymous mutations

• Mutations found in 73% of the patients and in 66% of

the panel genes

IRF4

STAT3

CCND1

CYLD

FAM46C

TP53

BRAF

TRAF3

DIS3

NRAS

KRAS

0% 5% 10% 15% 20% 25%

Top mutated genes

1 10 19 28 37 46 55 64 73 82 91 1001091181271361451541631721811901992082172262352442532622710%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

50% of the mutations

Var

iant

rea

d fr

eque

ncy

Mutations

Clonal diversity of the mutational spectrum

23%

<10%VR

<25%VR

Myeloma Specific Gene Mutation Panel Tracks Clonal Changes Over Time

Patient One Patient Two Patient Three

Var

ian

t re

ads

Number of patients (n=156) IncidenceKRAS 11111111111111111111111111111111111111

43%NRAS 111 1111111111111111111111

BRAF 1111 1111 111111

MAPK signaling pathway

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100% KRAS

NRAS

BRAF

status protein variant reads

untreated p.Gly466Glu 11%p.Gly469Arg 12%

untreated p.Gly466Ala 5%untreated p.Gly464Val 8%

treated p.Leu485Phe 31%untreated p.Lys499Asn 12%untreated p.Asp594Asn 18%untreated p.Asp594Gly 7%untreated p.Gly596Arg 29%untreated p.Val600Glu 14%untreated p.Val600Glu 44%untreated p.Val600Glu 12%untreated p.Val600Glu 25%untreated p.Val600Glu 5%untreated p.Val600Glu 23%

activating loop (AA594-601)phosphate binding loop (AA464-469)

BRAF mutations in 9% of patients• 1 patient with 2 BRAF mutations• 13 potentially activating mutations:

• 9 mutations in the activating loop, of which 6 at known actionable position V600E (4%)

• 4 more in the phosphate binding loop

Mutations in targetable genes include IDH1 R132 mutation

• Activating R132 IDH1 mutations are frequently found in

glioma, but also in other malignancies including acute

myeloid leukemia and have been described in myeloma.

• IDH1 inhibitors are in clinical investigation and

demonstrated clinical efficacy

• In our 14 advanced patients we identified 2 patients

(15%) with an IDH1 mutation (R132G and R132C)

Mutations in the Cereblon pathway in 5% of the patients

CRBN 1

CUL4B 1 1

IKZF3 1 1 1

IRF4 1 1 1 1 1

* = gene found mutated in cohort

• In an untreated patient refractory to Len/Dex induction therapy we identified mutations in CRBN and IRF4

• In a refractory patient progressed on Thalidomide, Lenalidomide and Pomalidomide IKZF3 was mutated.

• 4 out of 5 IRF4 mutated patients shared a K123R variant

IRF4MYC

MM cytotoxicity

CRBNIMiDs

IKZF1/3

*

*

*DDB1

CUL4

ROC1

*

Mutation in genes associated with proteasome inhibitor resistance

XBP1 (p.Leu232*)

Tumor

normal

• XBP1 needed for plasma cell maturation and suppression of XBP1s in MM has shown to induce bortezomib resistance

• In a patient that progressed on Carfilzomib we found a truncating XBP1 mutation

Leung-Hagesteijn et al, Cancer Cell 2013

• Clonal heterogeneity is prevalent

• Minor clones can be lethal

• Mutation profiling has potential to identify drug resistant clones and remission status in real time

• Clinical Markers of drug response are emerging

• IMiD: Cereblon, Ikaros, IRF4

• Proteasome: IRE1, XBP1

Summary

Thankyou: Comments, Questions or Insults ?