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`Amerigen Exhibit 1147
`Amerigen v. Janssen IPR2016-00286
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`
`

`
`REPRINT
`
`Journal of Chemotherapy
`
`Vol. 16 - Supplement n. 4 (7-12) - 2004
`
`REVIEW
`
`Reversing Resistance to Targeted Therapy
`
`L. VIDAL - G. ATTARD - S. KAYE - J. DE BONO*
`
`Institute of Cancer Research and Royal Marsden Hospital, Sutton, UK.
`
`* Correspondence: Dr. Johann S. de Bono, MB ChB, FRCP, MSc, PhD, Senior Lecturer and Consultant Medical
`Oncologist, Centre for Cancer Therapeutics, Institute for Cancer Research, Royal Marsden Hospital, Downs Road,
`Sutton, Surrey SM2 5PT, England. Tel: +44-20-8722-4028; Fax: +44-20-8642-7979. E-mail: jdebono@icr.ac.uk
`
`Summary
`
`The development of molecular targeted anticancer drugs is rapidly changing
`cancer therapeutics. However, drug resistance to these novel agents remains a real
`clinical concern. Reports now indicate that resistance to many of these molecular
`targeted agents - including hormone therapies, trastuzumab, imatinib, and gefitinib
`- occurs via common resistance mechanisms. These include 1) inadequate target
`blockade due to sub-optimal drug delivery; 2) altered target expression at the DNA
`(gene amplification), mRNA or protein level; 3) an altered target such as a mutated
`kinase domain; 4) modified target regulating proteins (e.g. altered expression of co-
`activators and/or co-repressors for nuclear steroid hormone receptors); 5) signal-
`ling by alternative proteins (functional redundancy) or different signalling pathways.
`It is envisioned that the molecular evaluation of clinical anticancer drug resistance,
`which requires the detailed study of pharmacokinetics, pharmacogenetics and phar-
`macodynamics, will allow the development of rational reversal strategies and impro-
`ved patient outcome.
`
`Key words: Targeted therapy, resistance.
`
`INTRODUCTION
`
`SPECIFIC MECHANISMS OF RESISTANCE
`
`Preferential cytotoxicity against malignant tissues
`remains tantamount to the Holy Grail in cancer
`therapeutics because this portends improved patient
`tolerance and quality of life and, importantly, the
`capacity to deliver combination therapy. Rationally
`designed and molecular target based anticancer
`agents are characterised by lower toxicity and wider
`therapeutic indices than traditional cytotoxic drugs.
`These agents may reverse or modulate chemothera-
`py resistance, enhance anti-tumour activity or main-
`tain tumour regression. This article reviews potential
`mechanisms of resistance to targeted therapies and
`suggests strategies to overcome this resistance,
`thereby maximizing anti-tumour effect and clinical
`benefit.
`
`Anti-androgen resistance
`
`Hormone therapy remains the mainstay for the
`treatment of metastatic prostate cancer. Androgen
`deprivation therapy induces a remission in 80 to
`90% of patients with advanced disease and results in
`a median progression-free survival of 12 to 33
`months, at which time an androgen-independent
`phenotype usually emerges. This accounts for the
`median overall survival of 23 to 37 months from the
`initiation of androgen deprivation 1. Various mecha-
`nisms of androgen resistance have been postulated.
`Inadequate LHRH and testicular androgen suppres-
`sion or circulating low levels of adrenal androgens
`can result in failure to respond to hormone therapy.
`Improving or increasing LHRH analogue delivery
`
`© E.S.I.F.T. srl - Firenze
`
`ISSN 1120-009X
`
`

`
`8
`
`L. VIDAL - G. ATTARD - S. KAYE - J. DE BONO
`
`can abrogate the former, while adrenal androgen
`synthesis can be inhibited by the administration of
`low doses of steroids or through the inhibition of
`key enzymes in the adrenal steroid biosynthesis
`pathways with agents such as ketoconazole or the
`CYP17 inhibitor abiraterone acetate 2. Preclinical
`data indicate that most androgen-independent
`prostate cancers continue to express androgen
`receptor (AR) with AR signalling remaining intact, as
`demonstrated by the expression of prostate-specific
`antigen (PSA), and consistently increased levels of
`AR mRNA, despite castrate testosterone levels 3.
`This could occur following binding of (low) levels of
`ligand to hypersensitive AR complexes or by andro-
`gen-independent activation of the AR. AR gene
`amplification resulting in increased receptor levels
`could also result in sensitisation of the cell to low lig-
`and concentrations. Similarly, point mutations of the
`AR gene may alter the antagonistic effect on the AR
`receptor seen with steroid ligands, such as oestro-
`gens, corticosteroids or even AR antagonists, to an
`agonistic one. Although these mutations appear to
`be uncommon in the clinic,4 they could explain the
`anti-androgen withdrawal syndrome observed in
`approximately 10-15% of hormone refractory
`prostate cancer (HRPC) patients 5.
`Hormone resistance in the majority of patients
`who do not have AR gene alterations but who retain
`active AR signalling may be explained by androgen-
`independent activation by growth factor signalling,
`which can directly activate the AR and its down-
`stream pathways. This may occur through activation
`of the erb-B receptors due to their aberrant expres-
`sion or the over-expression of their ligands 6. In
`addition, over-expression of insulin growth factor
`(IGF)-I and fibroblast growth factor (FGF)-8b has
`been reported in HRPC; binding to their respective
`receptors can bypass the AR and activate down-
`stream signalling 7,8. Ligand-independent activation
`of the AR can also occur through the increased
`expression of co-activators (such as SRC-1) or
`decreased expression of co-repressors (such as
`NCoR), which can alter AR transcriptional activity 9.
`Finally, alternative-signalling pathways involved in
`the regulation of apoptosis, such as the up-regula-
`tion of anti-apoptotic genes (e.g.Bcl-2 or the IAPs)
`could impart clinical androgen resistance. Strategies
`to reverse such resistance have been pursued in clin-
`ical trials utilising antisense technologies.
`
`ANTI-OESTROGEN RESISTANCE
`
`Intrinsic resistance or the development of
`acquired resistance to oestrogen manipulation
`remains a significant clinical problem in the treat-
`ment of oestrogen receptor (ER) positive breast can-
`cer 10. While up to 70% of patients with ER positive
`advanced disease initially respond to hormonal mod-
`ulation, most tumours will eventually become resis-
`
`tant. While the most important factor in defining
`hormonal resistance is lack of ER expression, the
`mechanisms of resistance in ER positive tumours
`have not been fully elucidated and may be multifac-
`torial.
`The ER pathway is complex and involves multi-
`ple co-regulating factors linked to other signalling
`pathways and in particular, is regulated through its
`phosphorylation on two serine residues which can
`be modulated by the PI3K/Akt and the Ras/Raf/Erk
`pathways. The ER forms multiprotein complexes
`with co-regulatory proteins that possess either his-
`tone acetylase or histone deacetylase activity. These
`co-regulators are recruited in the process of ER lig-
`and binding, increasing or blocking downstream
`effectors depending on the balance between co-acti-
`vators and co-repressors. Increased ER expression
`can alter the response of ER binding to ligands such
`as oestrogens or anti-oestrogens. Changes in host
`endocrinology, drug pharmacokinetics, altered α:β
`ER ratios, ER mutations, perturbations in down-
`stream growth factor signalling, and altered ER co-
`regulating factors have all been implicated in this
`resistance phenotype 10.
`Reports have described a mutation in the hor-
`mone-binding domain of ER-α that is hypersensitive
`to low levels of oestrogen compared with wild-type
`ER-alpha in preclinical models 11. This point muta-
`tion comprises an amino-acid substitution in the ER-
`α acetylation site 5 that alters the ability of the ER to
`bind to many co-regulatory factors and consequent-
`ly, its transcriptional activity. However, this mecha-
`nism of resistance remains controversial. Up-regula-
`tion of receptor co-activators is another possible
`mechanism of oestrogen resistance. Reports indicate
`that increased expression of co-activators may stimu-
`late growth in the absence of oestrogen 10. In this
`environment, tamoxifen may exert an agonistic
`rather than an antagonist effect. Furthermore, cross-
`talk between erb-B receptor signalling and the ER
`has been associated with endocrine therapy-resis-
`tance 10,12. The ER may be activated by phosphory-
`lation in the absence of its ligand oestradiol by
`growth factor signalling (e.g. EGF and IGF-1)
`through both mitogen-activated protein kinase and
`Akt pathways. Preclinical and clinical data have
`already demonstrated that epidermal growth factor
`receptor (EGFR) tyrosine kinase inhibitors may
`reverse resistance to tamoxifen, and clinical trials
`combining endocrine therapies and signal transduc-
`tion inhibitors are ongoing 12. Preliminary data from
`a phase II trial indicate that gefitinib is active in ER
`positive tamoxifen-resistant breast cancer patients
`13,14.
`Altered intracellular signalling, downstream of
`the receptor tyrosine kinases may also result in hor-
`mone resistance. This can be addressed by targeting
`downstream signal-transduction proteins that interact
`with the ER pathway; for example inhibiting Ras sig-
`nalling by the farnesyltransferase inhibitors (FTIs) or
`
`

`
`REVERSING RESISTANCE TO TARGETED THERAPY
`
`9
`
`the molecular target of rapamycin (mTOR) by the
`rapamycin analogues 15,16. Emerging clinical trial
`data indicate that these agents can reverse hormone
`resistance in advanced breast cancer.
`
`TRASTUZUMAB (HERCEPTIN™) RESISTANCE
`
`Many mechanisms may be involved in the devel-
`opment of resistance to trastuzumab. Redundant sig-
`nalling mediated by the hetero- or homo-dimeriza-
`tion of other members of the erb-B family (EGFR,
`HER3, HER4) may activate downstream signalling
`despite HER-2 blockade by trastuzumab 17.
`Trastuzumab resistance may be reversed by combin-
`ing agents that target different members of the erb-
`B family (for example, trastuzumab with gefitinib or
`trastuzumab with erlotinib) or using agents that tar-
`get more than one erb-B family member. Agents
`such as the human monoclonal antibody pertuzumab
`(Omnitarg/2C4) targeting the heterodimerization
`domain on HER2 or the pan-erbB small molecule
`inhibitors need to be evaluated in this setting.
`Trastuzumab resistance may also result from overex-
`pression of other growth factor receptors or their
`ligands (such as the IGF-1R) or altered downstream
`proteins (eg. loss of PTEN) that modulate down-
`stream signalling 18. Targeting downstream signalling
`with small molecule inhibitors such as the mTOR
`inhibitors, Raf kinase inhibitors or FTIs may there-
`fore reverse trastuzumab resistance. Finally, recent
`data suggest that some trastuzumab-resistant breast
`cancers express reduced p27kip1 levels with increased
`cdk2/4 activity, suggesting a role for drugs that
`induce p27kip1 protein expression or inhibit cdk2/4
`in the treatment of these patients 19. Small molecule
`cdk inhibitors are being evaluated in the clinic and
`may merit further evaluation in this setting.
`
`GEFITINIB RESISTANCE
`
`Gefinitib (IressaTM) is an EGFR tyrosine kinase
`inhibitor. Recent reports indicate that incomplete
`target blockade due to poor drug delivery may be a
`significant concern in the treatment of colorectal
`cancer 20. Tumour biopsies from 28 colorectal can-
`cer patients were collected before and after treat-
`ment with gefitinib (single agent gefitinib 250 mg vs
`500 mg on an ECOG randomised phase II trial
`comparing the two doses given continuously daily).
`EGFR, p-EGFR (tyrosine residue at position 1068),
`Akt, p-Akt, MAPK, p-MAPK and Ki67 were charac-
`terized 20. Only 3 tumours demonstrated a fall in p-
`EGFR after one week of gefitinib therapy while the
`remaining biopsies did not show p-EGFR inhibition
`indicating that gefitinib was not effectively inhibiting
`its target. No inhibition of downstream signalling or
`Ki67 expression was observed. In this study only 1
`of 110 patients treated had an objective response.
`
`Other reports indicate that patients who develop an
`acneiform skin rash while on treatment with an
`EGFR-targeting drug are more likely to show clinical
`benefit. This was noted in studies of patients with
`non-small cell lung cancer (NSCLC) treated with
`erlotinib and patients with colorectal carcinoma
`treated with cetuximab (ErbituxTM) 21. The develop-
`ment of skin rash reflects target blockade, suggesting
`that patients with higher drug exposure are more
`likely to respond. In an attempt to maximise target
`blockade and minimise inter-patient pharmacody-
`namic variability, it has been proposed that dosing
`with these agents should be increased until a grade
`1 or 2 (CTCAE) skin rash is observed.
`Incomplete target blockade is only one of several
`potential resistance mechanisms to treatment with
`EGFR inhibitors. Increased expression of the target
`EGFR or its ligands due to gene amplification,
`altered RNA stability or altered recycling can all
`result in gefitinib resistance. Mutations of the EGFR
`kinase domain can also result in altered EGFR sensi-
`tivity to gefitinib. The EGFRvIII mutation that results
`in amino-acid terminal truncation and constitutive
`activation of the receptor has been reported to be
`less sensitive to small molecule TKIs than the wild-
`type receptor. Moreover, mutations in threonine
`766 in the EGFR kinase domain confer resistance to
`quinazoline inhibitors 22. This site appears to be a
`“hot-spot” for the development of resistance since
`mutation of the corresponding threonines in C-Abl,
`p38 and Src also confer resistance to their respec-
`tive small molecule inhibitors 22. Conversely, data
`also indicate that pre-treatment EGFR mutations can
`increase sensitivity to EGFR blockade by gefitinib 23.
`Preliminary studies indicate that tumour samples
`from 9 NSCLC patients who responded to gefinitib
`were found to contain recurrent heterozygous single
`amino-acid mutations in the EGFR kinase domain (at
`exons 18, 19 and 21) in contrast to samples from
`the patients who did not respond that did not have
`any of these mutations. Mutated EGFRs were more
`sensitive to ligand stimulation and consequently,
`more sensitive to gefinitib.
`Inhibition of EGFR phosphorylation can also be
`uncoupled from inhibition of downstream signalling
`by alterations in other signalling effectors, such as
`constitutively activating mutations of Ras, B-Raf and
`PI3 kinase or loss of expression of the Akt regulator
`PTEN 24 or activation of other tyrosine kinase or G-
`protein coupled receptors (eg other erbB receptors
`or the IGF-IR). Combinations of targeted inhibitors
`need further evaluation in the clinic.
`
`IMATINIB RESISTANCE
`
`Imatinib (GlivecTM, GliveecTM), a small molecule
`inhibitor of BCR-ABL, C-kit and PDGFR-α, has had
`a major impact on the treatment of chronic myeloid
`leukaemia (CML) and gastrointestinal stromal
`
`

`
`10
`
`L. VIDAL - G. ATTARD - S. KAYE - J. DE BONO
`
`tumours (GIST). Several mechanisms of imatinib
`resistance have been reported. Incomplete target
`blockade due to inadequate drug delivery may be a
`reversible resistance mechanism. Reports indicate
`that in chronic phase, Philadelphia chromosome
`positive, CML higher doses of imatinib (600 or 800
`mg daily) may overcome resistance to lower doses
`(400 mg per day) 25. P-glycoprotein (mdr1) overex-
`pression has also been reported to confer resistance
`to imatinib in vitro by reducing intracellular imatinib
`concentrations, although these findings remain con-
`troversial since other reports indicate that imatinib is
`a poor substrate for p-glycoprotein 26,27. Other fac-
`tors that decrease imatinib delivery to its target
`include high levels of alpha-1 acid glycoprotein
`(AGP), which can prevent intracellular penetration
`by binding imatinib 28.
`Probably the most common mechanism of
`acquired imatinib resistance is alteration of the drug
`target 29-32. Gene amplification of BCR-ABL and
`mutations of key amino acids in the ABL or c-Kit
`kinase domains have been reported. Increasing drug
`dose, 25 or concurrently administering Hsp-90
`inhibitors that inhibit the expression of the mature
`target protein, may reverse the impact of BCR-ABL
`gene amplification 32. Approximately 30 different
`point mutations, coding for distinct single amino-acid
`substitutions in the BCR-ABL kinase domain, have
`been isolated from relapsed CML patients resistant
`to imatinib. While it has been suggested that some
`of these mutations predate imatinib therapy, in most
`cases the selective pressure of imatinib treatment
`induces these mutations. Many of these mutations
`have been shown to alter the imatinib-binding region
`of the BCR/ABL kinase domain, thus reducing ima-
`tinib’s binding affinity. These mutations occur either
`at sites that come into direct contact with imatinib
`(T315, F317, and F359), and presumably impair
`drug binding without significantly affecting the bind-
`ing of ATP, or at residues located in distant regions
`implicated in the unique conformational change that
`the kinase domain must undergo to accommodate
`imatinib 29. Crystallographic studies predict that most
`imatinib-resistant mutants should remain sensitive to
`inhibitors that bind ABL with less stringent confor-
`mational requirements than imatinib. Novel Abl
`kinase inhibitors are showing promising activity in
`preclinical studies and are in various stages of devel-
`opment 30. Conversely, however, it is important to
`note that in GIST activating mutations of c-KIT fre-
`quently predate therapy with imatinib and are key
`driving mutations in this disease, predicting response
`and correlating with clinical outcome with imatinib31.
`BCR-ABL signalling may also be inhibited
`through the induction of BCR-ABL protein degrada-
`tion using inhibitors of the molecular chaperone
`Hsp90 or the blockade of critical downstream signal
`transduction proteins 32,33. Hsp90 inhibitors, such as
`17-AAG, have potent BCR-ABL inhibitory activity
`and clinical trials with 17-AAG and imatinib in CML
`
`are underway. Preclinical studies also suggest that
`inhibition of farnesyltransferase, presumably through
`inhibition of Ras downstream signalling, may also
`reverse imatinib resistance 33.
`
`BEVACUZIMAB (AVASTIN™) RESISTANCE
`
`Angiogenesis inhibition has emerged as an
`important therapeutic strategy in the treatment of
`malignancy 34,35. Tumour cells recruit endothelial
`cells by secreting pro-angiogenic factors such as vas-
`cular endothelial growth factor (VEGF) and fibroblast
`growth factor (FGF). VEGF is overexpressed in
`many tumours and is associated with a worse clinical
`outcome. The aetiology of resistance to anti-angio-
`genesis agents has not yet been extensively
`explored. Nonetheless, similar mechanisms of resis-
`tance, as have been described for other targeted
`agents, are likely. Elevated pro-angiogenic ligand (eg
`VEGFs, FGFs) expression may contribute to antian-
`giogenesis drug resistance, and have already been
`reported to contribute to resistance to chemotherapy
`or endocrine therapy 35,36,37. It is plausible that resis-
`tance to bevacuzimab, a humanised monoclonal
`antibody to VEGF that in combination with
`chemotherapy has resulted in a survival advantage in
`colorectal cancer, may result from increased expres-
`sion of VEGF. This may be reversed by increasing
`bevacuzimab dosing. Rational combinations of
`angiogenesis inhibitors may optimise the antitumour
`effects of these therapeutics.
`
`CONCLUSION: REVERSING RESISTANCE
`TO TARGETED DRUGS
`
`Overall, a number of common resistance mecha-
`nisms have been reported for targeted therapeutics.
`These include 1) inadequate target blockade due to
`sub-optimal drug-delivery; 2) altered target expres-
`sion at the DNA (gene amplification), mRNA or pro-
`tein level; 3) an altered target such as a mutated
`kinase domain; 4) modified target regulating pro-
`teins; 5) signalling by alternative proteins (functional
`redundancy) or different signalling pathways. Several
`strategies for the reversal of resistance to targeted
`anticancer therapeutics can therefore be considered
`and may be widely applicable.
`The first consideration to reversal of resistance
`involves the evaluation of whether maximal target
`blockade has been achieved. Increasing drug dosing
`can reverse resistance, although this may be limited
`by toxicity. Increasing drug delivery by decreasing
`intratumoral interstitial pressure through the inhibi-
`tion of PDGF receptor signaling, may also reverse
`resistance 38. Emerging clinical data suggest that dif-
`ferences in the therapeutic efficacy between mono-
`clonal antibodies and small molecules targeting
`EGFR signaling in colorectal cancer may be due, at
`
`

`
`REVERSING RESISTANCE TO TARGETED THERAPY
`
`11
`
`least in part, to inadequate target blockade 20,39. The
`longer half-life of monoclonal antibodies and their
`irreversible target modulation may be a major advan-
`tage for these agents over the small molecule
`inhibitors. Increasing the frequency of administration
`of the small molecules may help circumvent this and
`result in more complete and sustained target inhibi-
`tion. Moreover, irreversible inhibitors may also
`achieve more profound target inhibition.
`An alternative therapeutic strategy for overcom-
`ing resistance to targeted therapeutics involves the
`inhibition of the post-translational maturation of the
`protein target by modulating the chaperone Hsp-90.
`Hsp90 inhibition modulates the expression of many
`proteins such as HER2, Bcr-Abl, HIF-1-alpha, ER,
`AR, Akt and Raf 40. Hsp90 inhibitors are therefore
`now being evaluated in clinical trials in combination
`with targeted therapies such as imatinib in order to
`reverse resistance to these agents.
`Gene mutations are common causes of acquired
`resistance to targeted therapies, usually changing the
`conformation of the tyrosine kinase-binding site.
`Using molecular and crystallography studies to study
`these alterations, novel compounds that can inhibit
`these mutated targets are being developed. Novel
`Bcr-Abl small molecule inhibitors that can inhibit
`imatinib refractory, mutated, Abl kinases are already
`being evaluated in the clinic 30.
`Finally, the highly selective inhibition of a molec-
`ular target may fail to translate into clinical benefit
`due to the existence of alternative proteins or path-
`ways that may abrogate the induction of cell death
`following target blockade, due to either functional
`redundancy or the complexity of the molecular alter-
`ations inherent to the cancer cell. Combinations of
`targeted therapeutics therefore need to be evaluated
`in the clinic in order to inhibit such escape mecha-
`nisms. Combinations of for example imatinib and
`the m-TOR inhibitor RAD-001 are being evaluated
`in imatinib resistant GIST; erlotinib and trastuzumab
`are being evaluated in combination in HER2 positive
`breast cancer as is the farnesyltransferase inhibitor
`SCH66336 with trastuzumab. The clinical evalua-
`tion of combinations of unapproved, investigational,
`targeted therapies remains, however, a major logisti-
`cal challenge since this frequently involves more
`than one industry partner. This has resulted in an
`increasing interest in less selective agents that can
`hit multiple targets. This strategy has proved suc-
`cessful in the treatment of renal cell carcinoma utilis-
`ing the small molecule inhibitor BAY 43-9006 in
`the treatment of clear cell renal cell carcinoma 41.
`BAY 43-9006 targets VEGFR2, Raf kinase, c-Kit
`and PDGFRβ. A combination-based strategy, utilis-
`ing the EGFR inhibitor erlotinib and the antibody to
`VEGF bevacuzimab, has also shown promising effi-
`cacy (both drugs are being developed by Genentech,
`CA) 42. The biology-based development of combina-
`tions of molecular targeted therapies is likely to be
`key in the future treatment of cancer 43.
`
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