Rational drug design and resistance in chronic myelogenous ... · the idea of rational anti-cancer...

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First studies seeking for resistance mechanisms found out that relapse was dependent on BCR-ABL. These initial experiments revealed two resistance mechanisms: The aim of this bibliographic revision is to describe the process of development of TKIs, specially emphasizing on the idea of rational anti-cancer drug design as the main strategy for overcoming resistance to therapy. I also pretend to review the actual situation of CML thanks to TKIs, an example of how biochemistry and molecular pathology can highly improve medicine. Data has been obtained using mainly the searching engine Pubmed. Original articles of key publications of each progress in the field have been used, as well as recent reviews to understand the current situation. The complex array of mutations observed rendered difficult to envision a single second generation TKI active against all the mutations. A significant number of mutations prevent the kinase domain from achieving the closed conformation necessary for drug binding. SOLUTION to find inhibitors that bind Abl in the open configuration or with less stringent conformational requirements. Once reviewed the development procedure of TKIs and their impact on CML patients, the following conclusions can be drawn: Imatinib and the rest of the TKIs approved for CML represent the first case of rational drug design against a human malignancy They also are an example of the problem that resistance supposes to cancer and a way of solving them Finally, CML is a pathology that can take profit from personalized medicine, owing to the different mutational profile of the different available drugs and the diversity profile of each one. Rational drug design and resistance in chronic myelogenous leukemia Helena Jover Escapa. Biomedical Sciences degree Universitat Autònoma de Barcelona Introduction Objectives and methodology From Philadelphia chromosome to Imatinib initial results Conclusions Current situation and treatment algorithm References Frontline therapy *: Imatinib (400mg daily ) Optimal response Continue therapy indefinitely (persistence of leukemic stem cells) Suboptimal response (CCyR at 12 months/ hematologic or cytogenetic relapse) Intolerance (adverse events usually bothering but non dangerous) Mutational analysis Switching of TKI Dasatinib (140mg daily) Nilotinib (400mg daily) twice Bosutinib (500mg daily) Ponatinib (45mg daily) MP: Y253H, E255K/V, or F359C/V/I TP: Myelosuppression (20–30%), particularly thrombocytopenia, and pleural (10–25%) or pericardial effusions (≤5%). Pulmonary hypertension (<1-2%). MP: V299L, T315A, F317L./F/I/7 TP: Hyperglycemia (10–20%), pruritus and skin rashes, headaches, pancreatitis (<5%). Vasospastic/vaso- occlusive events at low but significant rates. MP: most Imatinib resistant-mutants, except T315I and V299L. TP: Early and self-limited gastrointestinal complications, diahrrea (50-70%). MP: T315I (unique option) TP: Skin rashes (10–15%), pancreatitis (5%), elevations of amylase/lipase (10%), vasospastic/vaso-occlusive events (10-20%) and systemic hypertension. Possibility of treatment discontinuation in controlled clinical trials *Dasatinib and Nilotinib are also indicated as frontline therapy Consider dose escalation Abbreviations CCyR: complete cytogenetic response, no Ph+ cells detected by conventional or FISH cytogenetic testing. MR: molecular response, nevative PCR when testing for BCR-ABL in bone marrow cells of CML patients. TP: toxic profile MP: mutational profile If deep and sustained MR (≥2 years) Figure 1. Translocation that creates de Philadelphia chromosome 1 . Overcoming resistance Resistance mechanisms Figure 2. Responses of a group of six tyrosine kinase enzymes to Imatinib. The ability to inhibit c-Abl without affecting other enzymes is critical for therapy success 2 . 9 22 9q+ Ph 22q- Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that accounts for 15% of all cases of leukemia and has an annual incidence of 1.5 cases per 100,000 individuals. CML is driven by the BCR-ABL chimeric gene product, a constitutively active tyrosine kinase. The fusion gene results from the reciprocal balanced translocation t(9;22) (q34;q11.2), cytogenetically detected as the Philadelphia (Ph) chromosome [Fig.1]. The BCR-ABL oncoprotein possesses constitutive kinase activity that leads to the stimulation of cell-growth pathways and reduced apoptosis of CML cells. Examples of drivers implicated on the transduction pathways affected are RAS and MAP kinases. The natural course of CML consist on 3 phases: chronic phase, accelerated phase and a terminal blastic phase characterized by the typical acute leukemia symptoms. Before the era of selective BCR-ABL tyrosine kinase inhibitors (TKIs), the median survival at diagnosis in CML was 3–7 years. TKIs have revolutionized the treatment, natural history, and prognosis of CML. They have turned it in to the first cancer in which a medical treatment can return patients to a normal life expectancy. Gene amplification Multiple copies of the BCR-ABL gene were detected (by fluorescence in situ hybridization (FISH) in CML cells of patients that relapsed 4 . Point mutations After sequencing the region corresponding to the ATP binding pocket and the activation loop of the kinase domain of BCR-ABL of some CML patients, a single nucleotide change mutation (T315I) was seen to be among those that form part of a critical hydrogen bond with Imatinib 4 . Further studies proved that 90% of CML patients who relapsed after responding to Imatinib had different kinase domain mutations. To gain insight into the mutation mechanisms, they modelled each aminoacid substitution onto the crystal structure of the ABL kinase domain bound to Imatinib. This allowed the classification of mutations into two groups: Mutations that directly contact Imatinib (1-3). Mutations that prevent the conformation required for Imatinib-binding. These include mutations in the P loop region ATP phosphate binding loop (4-8) and in the vicinity of the activation loop (9-13). Figure 5. ABL kinase domain bound to Imatinib with 13 resistance mutations 5 . Despite their efficacy and optimal safety profile, all these TKI are ineffective to the T315I mutation. T315 residue is located on the gatekeeper region of the ATP-binding site. It participates in a critical hydrogen bonding interaction required for high-affinity binding of the other TKIs. The T315I mutation alters the topology of the ATP-binding pocket causing a steric clash between the side chain of the isoleucine and the hydrogen from the drug. SOLUTION Structural-guided experiments of an inhibitor that accommodates into the T315I side chain thanks to a carbon-carbon triple bond linkage on its structure. It’s the case of Ponatinib, a 500-fold potent than Imatinib approved by FDA in 2012. Philadelphia chromosome is proposed as the cause of CML 1987 BCR-ABL protein identified as cause of CML 1996 Screening of multiple compounds searching for a kinase small molecule inhibitor Druke and Lydon come up with the idea of blocking BCR-ABL to kill cancer cells, constituting the first hypothesis of a molecularly targeted antitumor therapy After various chemical improvements on a phenylaminopyrimidine class of inhibitors, they eventually gave rise to Imatinib 1998 1993 Fase I/II clinical trial with a ~100% of complete hematologic response 2001 FDA approval of Imatinib / Expansion into other malignancies / Important proportion of relapse observed in patients in advanced or blastic phase 2002 Figure 3. Overall survival in patients with CML treated with Imatinib, interferon or conventional chemotherapy 3 . BCR-ABL and Imatinib 3D structure elucidated [Fig. 4] / Studies to understand resistance mechanisms 1960 1988 Figure 4. Imatinib targets the relatively well conserved ATP-binding pocket of the catalytic domain of Abl, but it can still achieve high specificity since it recognizes a distinctive inactive conformation of the activation loop of Abl, which is seen to differ from the inactive conformations of other kinases. These characteristics give both high specificity and affinity to Imatinib 2 . DASATINIB (2006*) NILOTINIB (2007) BOSUTINIB (2012) Dual-specific Src/Abl kinase Binds BCR-ABL in both the active and inactive conformations [Fig. 8] Active against 14/15 Imatinib- resistant BCR-ABL mutants More than 300-fold potent than Imatinib Imatinib analogue, subtle differences account for greater potency [Fig. 7] Active against 32/33 Imatinib- resistant BCR-ABL mutants [Fig. 6] Less activity on the usual off- targets of Imatinib: c-Kit and PDGFR receptors. Dual-specific Src/ Abl kinase Activity against most Imatinib-resistant mutants of BCR-ABL Minimal inhibitory activity against c-Kit and PDGF receptors *Year of FDA approval Figure 7. Binding modes of the Abl inhibitors Imatinib, Nilotinib and Dasatinib. Positions of the P-loop and activating loop vary according to whether the kinase is in an active conformation. The green helix is helix C, which often moves between the active and inactive states of kinases 6 . Figure 6. Abl binded to Nilotinib. The locations of the amino acid substitutions of carried by the Imatinib-resistant BCR-ABL proteins are indicated in red, orange or green, and show different levels of sensibity to Nilotinib. T315I mutation is also resistant to Nilotinib 2 . Figure 8. The triple bond is an unique structural feature of Ponatinib that allows to evade the mutant gatekeeper residue I315 7 . After 15 years of clinical use of Imatinib and thanks to the other TKI available, CML has become the first cancer in which a medical treatment can return patients to a normal life expectancy. In spite of superior data, since neither Dasatinib or Nilotinib have shown substantial amelioration in either overall survival (OS) or progression free survival (PFS) rates over Imatinib, it continues to represent the most commonly used TKI to treat CML frontline Moreover, owing to some important risks of second generation TKIs shown in the table, it may not be worth it to take them if there is no resistance or intolerance to Imatinib. 1. Lydon N. Attacking cancer at its foundation. Nat Med. 2009; 15(10):1153-57. 2. Weinberg RA. The biology of cancer. Vol. 2 nd ed. New York: Garland Science; 2014. 3. Druker BJ. Perspectives on the development of imatinib and the future of cancer research. Nat Med. 2009; 15(10):1149-52. 4. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 2001; 293(5531):876–80. 5. Sawyers CL. Shifting paradigms: the seeds of oncogene addiction. Nat Med. 2009; 15(10):1158-1161 6. Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin D. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukemia. Nat Rev Cancer. 2006;7:345-56 7. Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I, et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med. 2012;367(22):2075–88.

Transcript of Rational drug design and resistance in chronic myelogenous ... · the idea of rational anti-cancer...

Page 1: Rational drug design and resistance in chronic myelogenous ... · the idea of rational anti-cancer drug design as the main strategy for overcoming resistance to therapy. I also pretend

First studies seeking for resistance mechanisms found out that relapse was dependent onBCR-ABL. These initial experiments revealed two resistance mechanisms:

The aim of this bibliographic revision is to describe theprocess of development of TKIs, specially emphasizing onthe idea of rational anti-cancer drug design as the mainstrategy for overcoming resistance to therapy. I also pretendto review the actual situation of CML thanks to TKIs, anexample of how biochemistry and molecular pathology canhighly improve medicine.

Data has been obtained using mainly the searching enginePubmed. Original articles of key publications of eachprogress in the field have been used, as well as recentreviews to understand the current situation.

The complex array of mutations observed rendered difficult to envision a single second generation TKI activeagainst all the mutations.

A significant number of mutations prevent the kinase domain from achieving the closed conformationnecessary for drug binding.

SOLUTION to find inhibitors that bind Abl in the open configuration or with less stringent conformationalrequirements.

Once reviewed the development procedure of TKIs and their impact on CML patients, the following conclusions can be drawn: Imatinib and the rest of the TKIs approved for CML represent the first case of rational drug design against a human

malignancy They also are an example of the problem that resistance supposes to cancer and a way of solving them Finally, CML is a pathology that can take profit from personalized medicine, owing to the different mutational profile of the

different available drugs and the diversity profile of each one.

Rational drug design and resistance in chronic myelogenous leukemia

Helena Jover Escapa. Biomedical Sciences degreeUniversitat Autònoma de Barcelona

Introduction Objectives and methodology

From Philadelphia chromosome to Imatinib initial results

Conclusions

Current situation and treatment algorithm

References

Frontlinetherapy*:Imatinib

(400mg daily)

Optimalresponse

Continue therapy indefinitely

(persistence of leukemic stem cells)

Suboptimalresponse

(CCyR at 12 months/hematologic or

cytogenetic relapse)

Intolerance(adverse events

usually bothering but non dangerous)

Mutational analysis

Switching of TKI

Dasatinib(140mg daily)

Nilotinib(400mg daily)

twice

Bosutinib(500mg daily)

Ponatinib(45mg daily)

MP: Y253H, E255K/V, or F359C/V/ITP: Myelosuppression (20–30%),particularly thrombocytopenia, andpleural (10–25%) or pericardialeffusions (≤5%). Pulmonaryhypertension (<1-2%).

MP: V299L, T315A, F317L./F/I/7TP: Hyperglycemia (10–20%),pruritus and skin rashes, headaches,pancreatitis (<5%). Vasospastic/vaso-occlusive events at low but significantrates.

MP: most Imatinib resistant-mutants,except T315I and V299L.TP: Early and self-limitedgastrointestinal complications,diahrrea (50-70%).

MP: T315I (unique option)TP: Skin rashes (10–15%),pancreatitis (5%), elevations ofamylase/lipase (10%),vasospastic/vaso-occlusive events(10-20%) and systemic hypertension.

Possibility of treatmentdiscontinuation in controlledclinical trials

*Dasatinib andNilotinib arealso indicated asfrontlinetherapy

Consider doseescalation

AbbreviationsCCyR: complete cytogenetic response, no Ph+ cellsdetected by conventional or FISH cytogenetictesting.MR: molecular response, nevative PCR when testingfor BCR-ABL in bone marrow cells of CML patients.TP: toxic profileMP: mutational profile

If deep and sustained MR (≥2 years)

Figure 1. Translocation that creates de Philadelphia chromosome1.

Overcoming resistance

Resistance mechanisms

Figure 2. Responsesof a group of sixtyrosine kinaseenzymes toImatinib. The abilityto inhibit c-Ablwithout affectingother enzymes iscritical for therapysuccess2.

9 22

9q+

Ph 22q-

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that accounts for 15% of all cases of leukemia and has an annualincidence of 1.5 cases per 100,000 individuals.

CML is driven by the BCR-ABL chimeric gene product, a constitutively active tyrosine kinase. The fusion gene results from thereciprocal balanced translocation t(9;22) (q34;q11.2), cytogenetically detected as the Philadelphia (Ph) chromosome [Fig.1].

The BCR-ABL oncoprotein possesses constitutive kinase activity that leads to the stimulation of cell-growth pathways and reducedapoptosis of CML cells. Examples of drivers implicated on the transduction pathways affected are RAS and MAP kinases.

The natural course of CML consist on 3 phases: chronic phase, accelerated phase and a terminal blastic phase characterized by thetypical acute leukemia symptoms.

Before the era of selective BCR-ABL tyrosine kinase inhibitors (TKIs), the median survival at diagnosis in CML was 3–7 years. TKIs haverevolutionized the treatment, natural history, and prognosis of CML. They have turned it in to the first cancer in which a medicaltreatment can return patients to a normal life expectancy.

Gene amplificationMultiple copies of the BCR-ABL gene weredetected (by fluorescence in situ hybridization(FISH) in CML cells of patients that relapsed4.

Point mutationsAfter sequencing the region corresponding to the ATPbinding pocket and the activation loop of the kinasedomain of BCR-ABL of some CML patients, a singlenucleotide change mutation (T315I) was seen to beamong those that form part of a critical hydrogen bondwith Imatinib4.

Further studies proved that 90% of CML patients who relapsed afterresponding to Imatinib had different kinase domain mutations.

To gain insight into the mutation mechanisms, they modelled eachaminoacid substitution onto the crystal structure of the ABL kinase domainbound to Imatinib. This allowed the classification of mutations into twogroups:

Mutations that directly contact Imatinib (1-3).

Mutations that prevent the conformation required for Imatinib-binding.These include mutations in the P loop region ATP phosphate binding loop(4-8) and in the vicinity of the activation loop (9-13).

Figure 5. ABL kinase domainbound to Imatinib with 13resistance mutations5.

Despite their efficacy and optimal safety profile, all these TKI are ineffective to the T315I mutation.

T315 residue is located on the gatekeeper region of the ATP-binding site. It participates in a critical hydrogenbonding interaction required for high-affinity binding of the other TKIs. The T315I mutation alters thetopology of the ATP-binding pocket causing a steric clash between the side chain of the isoleucine and thehydrogen from the drug.

SOLUTION Structural-guided experiments of an inhibitor that accommodates into the T315I side chain thanksto a carbon-carbon triple bond linkage on its structure. It’s the case of Ponatinib, a 500-fold potent than Imatinibapproved by FDA in 2012.

Philadelphia chromosome is proposed as the cause of CML

1987

BCR-ABL proteinidentified as cause of CML

1996

Screening of multiple compoundssearching for a kinase smallmolecule inhibitor

Druke and Lydon comeup with the idea ofblocking BCR-ABL to killcancer cells, constitutingthe first hypothesis of amolecularly targetedantitumor therapy

After various chemical improvements on a phenylaminopyrimidineclass of inhibitors, they eventually gave rise to Imatinib

19981993

Fase I/II clinical trial with a ~100% of complete hematologic response

2001

FDA approval of Imatinib / Expansion into other malignancies / Important proportion of relapse observed in patients in advanced or blastic phase

2002

Figure 3. Overallsurvival inpatients withCML treatedwith Imatinib,interferon orconventionalchemotherapy3.

BCR-ABL and Imatinib 3D structure elucidated [Fig. 4]/ Studies to understand resistance mechanisms

19601988 Figure 4. Imatinib targets the relatively

well conserved ATP-binding pocket of thecatalytic domain of Abl, but it can stillachieve high specificity since itrecognizes a distinctive inactiveconformation of the activation loop ofAbl, which is seen to differ from theinactive conformations of other kinases.These characteristics give both highspecificity and affinity to Imatinib2.

DASATINIB (2006*) NILOTINIB (2007) BOSUTINIB (2012)

• Dual-specific Src/Abl kinase

• Binds BCR-ABL in both theactive and inactiveconformations [Fig. 8]

• Active against 14/15 Imatinib-resistant BCR-ABL mutants

• More than 300-fold potentthan Imatinib

• Imatinib analogue, subtledifferences account for greaterpotency [Fig. 7]

• Active against 32/33 Imatinib-resistant BCR-ABL mutants [Fig. 6]

• Less activity on the usual off-targets of Imatinib: c-Kit andPDGFR receptors.

• Dual-specific Src/Ablkinase

• Activity against mostImatinib-resistantmutants of BCR-ABL

• Minimal inhibitory activityagainst c-Kit and PDGFreceptors

*Year of FDA approval

Figure 7. Binding modes of the Ablinhibitors Imatinib, Nilotinib andDasatinib. Positions of the P-loop andactivating loop vary according towhether the kinase is in an activeconformation. The green helix is helixC, which often moves between theactive and inactive states of kinases6.

Figure 6. Abl binded to Nilotinib. Thelocations of the amino acidsubstitutions of carried by theImatinib-resistant BCR-ABL proteinsare indicated in red, orange or green,and show different levels of sensibityto Nilotinib. T315I mutation is alsoresistant to Nilotinib2.

Figure 8. The triple bond is an uniquestructural feature of Ponatinib thatallows to evade the mutant gatekeeperresidue I3157.

After 15 years of clinical use of Imatinib and thanks to the other TKI available, CML has become thefirst cancer in which a medical treatment can return patients to a normal life expectancy.

In spite of superior data, since neither Dasatinib or Nilotinib have shown substantial amelioration ineither overall survival (OS) or progression free survival (PFS) rates over Imatinib, it continues torepresent the most commonly used TKI to treat CML frontline

Moreover, owing to some important risks of second generation TKIs shown in the table, it may notbe worth it to take them if there is no resistance or intolerance to Imatinib.

1. Lydon N. Attacking cancer at its foundation. Nat Med. 2009; 15(10):1153-57.2. Weinberg RA. The biology of cancer. Vol. 2nd ed. New York: Garland Science; 2014.3. Druker BJ. Perspectives on the development of imatinib and the future of cancer research. Nat Med. 2009; 15(10):1149-52.4. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 2001;

293(5531):876–80.5. Sawyers CL. Shifting paradigms: the seeds of oncogene addiction. Nat Med. 2009; 15(10):1158-11616. Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin D. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukemia. Nat Rev Cancer.

2006;7:345-567. Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I, et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med. 2012;367(22):2075–88.