Resistance in the Clinical Setting Dr. Wilson H. Miller, Jr

Resistance in the Clinical Setting Dr. Wilson H. Miller, Jr

Potential Conflict of Interest

Wilson H. Miller Jr., M.D., Ph.D.Wilson H. Miller Jr., M.D., Ph.D.

Segal Cancer CenterSegal Cancer CenterSMBD Jewish General HospitalSMBD Jewish General HospitalMcGill University, Montreal, Quebec CanadaMcGill University, Montreal, Quebec Canada

Resistance in the Clinical SettingResistance in the Clinical Setting

Mechanisms of Cellular Drug Resistance

Intrinsic Resistance Mechanisms

Host factors•Decreased intracellular drug accumulation (poor absorption, rapid metabolism, or excretion).

• Inefficient delivery of a drug to its target (tumor cells).

Specific genetic and epigenetic drivers•Malignant cell growth is associated with tumor-specific activation of oncogenic pathways and inactivation of tumor suppressor genes.

•Specific drug targets may or may not be relevant to growth of a given tumor.

The wrong target cell? •Stem cell resistance

Mechanisms of Cellular Drug Resistance

Acquired Resistance Mechanisms

Decreased accumulation of drugs within cells• Increased drug efflux.• Reduced drug uptake.

Changes in drug-target interactions• Mutations in targeted oncogenes.• Changes in target gene expression.

Changes in signaling pathways that drive growth• Replacement of one TK pathway with another.• Interchangeable pro-angiogenic factors and pathways.• Multiple interdependent cell survival pathways.• Loss of checkpoints.

(1) Decrease in intracellular drug concentrations

(2) Changes in drug-targetinteractions

(3) Changes in signal transduction pathways Cell cycle arrest and repair

Three Main Mechanisms of Cellular Drug Resistance

Mutation

Decrease of drug influx• Alterations of cell membrane structures.• Most chemotherapeutic drugs enter cells by passive diffusion.

Increase of drug efflux• Overexpression of transmembrane proteins (ABC superfamily of

transporters).

Decrease in Intracellular Drug Concentrations

LRP/MVPIs the major component of the Vault protein Involved in cellulartraffic

Examples of Chemotherapeutic Drugs with Increased Delivery to Tumors

SarCNU RationaleSarCNU Rationale• SarCNU is a novel chloroethylnitrosourea which SarCNU is a novel chloroethylnitrosourea which

demonstrates selective cytotoxicity against primary demonstrates selective cytotoxicity against primary human gliomas in-vitro.human gliomas in-vitro.

• Selective uptake via the extraneuronal catecholamine Selective uptake via the extraneuronal catecholamine uptake carrier allows increased concentration in uptake carrier allows increased concentration in tumor cells.tumor cells.

• Preclinical toxicity studies confirm that SarCNU is Preclinical toxicity studies confirm that SarCNU is less toxic than BCNU, the standard treatment of less toxic than BCNU, the standard treatment of gliomas.gliomas.

SarCNUSarCNU

• Phase I and pharmacokinetics study in advanced Phase I and pharmacokinetics study in advanced solid tumor malignancy.solid tumor malignancy.

• 43 patients enrolled.43 patients enrolled.

• Myelosuppression and some pulmonary toxicity Myelosuppression and some pulmonary toxicity observed in patients.observed in patients.


Darinaparsin: Organic Arsenic

• First in a new class of molecules.• Potentially safer and more active for cancer treatment

than approved inorganic arsenic.

OH

HN

O

NH

NH2

OS

AsCH3H3C

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Darinaparsin (DAR) is more potent than As2O3 at inducing apoptosis in a variety of leukemia and lymphoma cell lines.

NB4 (APL) AsR2 (As-resistant APL)

IM9 (NHL)CCRF-CEM (NHL)

Diaz et al, 2009 Feb;23(2):431

DAR induces more cellular oxidative stress than As2O3. NB4 (APL) AsR2 (APL)

NB4 (APL) AsR2 (APL)

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Diaz et al, 2009 Feb;23(2):431

NB4 (APL) AsR2 (APL)

Increased ABCC1 exporter expression causes resistance to As2O3 but not DAR in the arsenic-resistant cell line AsR2.

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ATO-resistant NB4-AR2 cells, are sensitized to ATO, but not DAR, by co-treatment with an ABCC1 inhibitor.

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Total intracellular As in AsR2 treated for 24hrs.

Viable cell number in AsR2 treated for 24hrs.

RN1

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Figure 2. Chemical structure of RN1 and it’s precursors.

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Hybrid Molecules – Targeting the Oncogene with Two Therapeutic Agents


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0)NB4 and R4 cells were treated with media, 10-5 M RA, butyrate, RA plus butyrate, or RN1. In NB4 cells, RA, RA plus butyrate, and RN1 significantly inhibited growth (P<0.001). In R4 cells, RN1 significantly inhibited growth (P<0.02).

RN1 induces growth arrest in NB4 and R4 cell lines.

NB4 R4

Imatinib Treatment in CML

Chronic Myeloid Leukemia (CML)

• Characterized by the Philadelphia chromosome t(9;22).

• Results in fusion of BCR and ABL genes.

• Imatinib mesylate is the frontline therapy.

• Imatinib is a selective inhibitor of Bcr-Abl, PDGF-R, Kit.

Imatinib Treatment in CML Models Multiple Resistance Mechanisms

Primary resistance

• Insufficient inhibition of BCR-ABL • Low plasma levels of imatinib.• Activity of drug pumps.• Stem cells

Secondary resistance• Imatinib-resistant BCR-ABL kinase-domain mutations.• Overproduction of BCR-ABL (genomic amplification).• BCR-ABL-independent mechanisms (not well understood).

• ? Activation of other kinases.• ? Other molecular events.

Imatinib has revolutionized treatment for CML but resistance is a problem in a small percentage of patients.

BCR-ABL Mutations Associated with Imatinib Resistance

F486S F486S E255K/V/V

M244V M351T/L/L

M343T M343T

Y253F/H

E279K E279K

F317L

E355G/D/D

F359V/C/D/I/C/D/I

H396R/P/P

Q252H/R/R

S417YS417Y

E459K/Q E459K/Q

E450G/Q/K E450G/Q/K

M388L M388L G250E/A/F /A/F

D276GD276GT277A/NT277A/N

L387F/M L387F/M

V379I V379I

A397PA397P

P-loopP-loop Activation loopActivation loop

T315I**F311L/I/VF311L/I/V

V289A/IV289A/I

L248V L248V

F382LF382L

E281A E281A

V299LV299L

L364I L364I

G383DG383D

L298VL298V

E292VE292V E453G/K/A/V E453G/K/A/V

Q447RQ447R

S438CS438C

G236E G236E

D241GD241G

M237IM237I

L324Q L324Q

K357RK357R

K285NK285N

E275KE275K

S348LS348LA344VA344V

A350VA350VM472IM472I

I418VI418V

Most mutated clones, except for T315I, may be eradicated with appropriate choice and combination among the second generation Abl TKIs (Dasatinib, Nilotinib, Bosutinib).

CML Stem Cells – Resistance to TKI’sPersistence of minimal residual disease

Possible mechanisms of stem cell resistance• High levels of ABC drug transporters.• Increased capacity for DNA repair.• Accumulation of mutations.• Quiescence.

Therapeutic Approaches for Stem Cell Resistance• Targeting the ABC transporters.• Targeting the different surface markers.• Targeting the pathways in stem cell renewal.• Targeting the quiescence.

Resistance in Signal Transduction Pathways – HER2 (ERBB2)

HER2 (ERBB2) Driven Breast Cancer

• Overexpression of the Her2 (ErbB2) protein found in 18-20% of breast tumors.• Correlates with more aggressive tumors.

Current targeted therapies

Trastuzumab (Herceptin) – monoclonal Ab specifically targets Her2.• Treatment for early stage HER2+ breast cancer.

•Resistance in vast majority of patients occur within 1 year.•HER2 mutations not commonly found.

Lapatinib -TKI inhibitor•Inhibits Her2 and EGFR.

Current Therapies to Overcome Trastuzumab Resistance

Lapatinib -TKI inhibitor• This combined inhibition can overcome Herceptin resistance in some cases.

LBH589 – Deacetylase inhibitor• Induces degradation of Her2, ER and pAKT.

• Phase Ib/IIa LBH589 in combo with Trastuzumab for HER2+ metastatic breast cancer.

• Enhances Her2 inhibition in combo with Trastuzumab or Lapatinib

Resistance in Signal Transduction Pathways:The importance of KRAS, BRAF and EGFR mutations in

EGFR signaling in Colon Cancer

Ligand binding to EGFRpromotes heterodimerization, activation and downstream pathways;

• Ras-Raf• MAPK• PI3K-Akt

•Ab against EGFR (Cetuximab and Panitumumab) inhibit downstream

pathways.

•Mutated KRAS or BRAF leads to

constitutive activated pathway.

•Mutated KRAS (~30% pts)

•Mutated BRAF (~10% pts)

•Cetuximab and Panitumumab

Only effective in KRAS and BRAF

wild type tumors.

The importance of KRAS status inMetastatic Colorectal Cancer

Response to Cetuximab According to the Presence or Absence of KRAS Mutation in the Overall 114 Patients

Tumor Response

KRAS mutation Wild type KRASPNo. of Patients % No. of Patients %

CR 0 0 2 2.6 < .001

PR 0 0 32 41.0

SD 14 38.9 26 33.3

PD 22 61.1 18 23.1

Total 36 100 78 100

Lievre, A. et al. J Clin Oncol; 26:374-379 2008

Copyright © American Society of Clinical Oncology

Lievre, A. et al. J Clin Oncol; 26:374-379 2008

(A) Progression-free survival (B) overall survival according to the presence or absence of KRAS mutation

PFS32 vs. 9 weeksP = 0.0000001

OS 14.3 vs. 10.1 monthsP = 0.0017

Signal Transduction Pathways:The importance of KRAS, BRAF and EGFR mutations in EGFR

signaling in lung adenocarcinoma

Oncogene mutations in the EGFR pathway in lung adenocarcinoma

• About 50% of lung adenocarcinoma harbor somatic mutations of six genes that encode proteins in the EGFR signaling pathway:

– KRAS mutations – EGFR mutations – Her-2 mutations– Her-4 mutations– BRAF mutations– Phosphatidylinositol 3-kinase (PI3K) mutations

KRAS mutations in lung adenocarcinoma

• KRAS mutation in 30% of lung adenocarcinoma.

• Association with smoking. Poor prognostic factor in resected tumors.

• Lack of sensitivity of KRAS mutated tumors to geftinib or erlotinib (EGFR inhibitors).

Activating Mutations in the EGFR Correlate with EGFR-TKI Sensitivity

EGFR mutations in lung adenocarcinomaassociated with sensitivity but additional mutations can

mediate resistance

Sharma, Nat Rev Cancer, 2007

Resistance in Angiogenic Targeted Therapy

Current Angiogenic Inhibitors in Clinical Use and Clinical Trials

• Bevacizumab (Avastin)• Sunitinib (Sutent) • Sorafenib (Nexavar)• Cederanib (Recentin - AZD- 2171)• VEGF-Trap

Many others in development

Upregulation of pro-angiogenic signaling pathways • FGF, ephrin and angiopoietin families.

Recruitment of BM derived cells•Endothelial and pericyte progenitors are incorporated as components of new vessels to build new blood vessels•Pro-angiogenic monocytes fuel the tumors with cytokines, growth factors and proteases.

Increased pericyte coverage protects tumor blood vessels • Helps tumor endothelium to survive and function.

Modes of Resistance to Anti-Angiogenic Therapy

Overcoming Resistance to Anti-Angiogenic Therapy

• The combination of antiangiogenesis agents with cytotoxic chemotherapy has increased the activity of chemotherapy in breast, colon, lung cancer and in melanoma.

• Data on toxicity of targeted agents in older individuals are limited: the risk of thrombosis with avastin and of serious cutaneous reactions with cetuximab appears to increase with age.

Overcoming Resistance

• Targeted therapy has been very successful in situations where a single or few targets are responsible to maintain the disease (CML, HER2 positive breast cancer).

• Inhibiting a single target in a complex signaling pathway is unlikely to provide sufficient therapeutic activity for the treatment of most genetically unstable human cancers.human cancers.

-Multiple activating signals and cross talk.-Multiple activating signals and cross talk.

-Signals transmitted via multiple pathways.-Signals transmitted via multiple pathways.

• The combination of 2 or more targeting agents seems to be more effective and safer, at least in the case of inhibition of the signal transduction cascade.

Conclusions

•Need to continue to characterize mechanisms of action, mechanisms of resistance, signaling pathways.

•Continued research to improve our understanding of the heterogeneity and complexity of the tumor microenvironment.

•Continue to identify mutations in targeted oncogenes and targets in the downstream pathways.

•The use of technological advances in genomics, proteomics and biomarker development to better predict tumor types and patient subsets that may be particularly responsive to treatment.

Conclusions: More work needed

Anti-estrogens ER, PR

Trastuzumab Her2 FISH, IHC

EGFR inhibitors ?FISH, ?IHC, mutation status

Anti-VEGF agents ??

PI3K-Akt-mTOR ??

IGF-I receptor antagonists ??

Src inhibitors ??

Cdk/Cyclin D1 inhibitors ??

HDAC/DNMT inhibitors ??

The Importance of Pharmacodynamic Markers

Preclinical

ClinicalSamples

Gene expressionGene expression

Enzyme activityEnzyme activity

Tumor cell markersTumor cell markers

•Data processing•Data integration•Pathway linkage•Analysis •Data coherence

•Data processing•Data integration•Pathway linkage•Analysis •Data coherence

BIOMARKERS

BIOMARKERS

Experiments Analysis Informatics Discovery

MetabolomicsMetabolomics

Translational research should be part of the solution

The complexity of resistance in patients demonstrates the need for• Developing new models of

– Multi-disciplinary and multi-institutional collaborations– Academic and industrial partnerships

• Designing biomarker-driven clinical trials to– Collect clinical samples– Identify biomarkers predicting resistance– Study mechanism of resistance identified in patients (vs. in cell lines)

– Develop new or improved molecules

The Quebec – Clinical Research Organization in Cancer was designed to answer these challenges.

Documents

Resistance in the Clinical Setting Dr. Wilson H. Miller, Jr