The BCR-ABL1 Gene

CML Pathogenesis-600pxBCR-ABL1 is a fusion gene formed as a result of the t(9;22)(q34;q11) chromosomal translocation, the translocation that results in the formation of the Philadelphia chromosome. The Abelson murine leukaemia viral oncogene homolog 1 (ABL1) gene from 9q34 is translocated downstream to a region at 22q11 known as breakpoint cluster (BCR). The fusion gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the expression chronic myeloid leukaemia phenotype. BCR-ABL1 gene has also been on implicated in the pathogens is of acute lymphoblastic leukaemia and in rare cases of acute myeloid leukaemia. The gene has been targeted with unparalleled success by the first tyrosine kinase inhibitor approved in clinical practice, imatinib.

 Molecular Biology of BCR-ABL

The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocated downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. ABL1-BCR translocation also occurs and may express but is of no clinical significance.


Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

The breakpoint of the ABL1 gene may be upstream exon 1a, between exon 1a and 1b or downstream exon 1b but it is almost always upstream exon 2. With rare exceptions all transcripts of BCR1-ABL1 gene have exon 2-11 of the ABL1 gene. The BCR breakpoints are variable and determine the size as well as the pathogenic properties of the BCR-ABL1 gene.. The breakpoint on the BCR gene are clustered in three regions known major cluster, minor cluster and micro cluster (Table 1). Depending on the location of breakpoint  on the BCR gene three types of protein are synthesized. The p210 transcript is associated with CML and some patients with Philadelphia positive acute lymphoblastic leukaemia. The shorter p190 transcript is associated with philedelphia positive acute lymphoblastic leukaemia and some patients of chronic myeloid leukaemia. The CML that carry this mutation show monocytosis and have a more aggressive course. The p230 is the largest and the rarest of the BCR-ABL1 transcripts. It is associated with a more indolent course and is found in patients with the rare chronic neutrophilic leukaemia. Atypical transcripts e1a3, e13a3 and e6a2 have been described.

Table 1: The BCR-ABL1 fusion genes

 Major Cluster  Minor Cluster  Micro-Cluster
 Synonym M-Cluster m-cluster µ-cluster
Location exons 12-16 Between alternative exon 2, e2’ and e2 between exons e19 and e20
Protein p210 p190 p230
Associated Leukaemia  CML, e14a2 shown to have thrombocytosis in some studies., Ph+ ALL  Ph + ALL; CML that tends to have monocytosis and an agressive course  Chronic Neutrophilic Leukaemia, Small reports describing patients with a course resembling classical CML

Mutagenicity of BCR-ABL1

ABL1 is a nuclear kinase whose activity is tightly regulated by the cell. BCR-ABL1 translocation results loss of regulation and the kinase is  cosntitutively active. Sustitution of ABL1 at the N termnal by segments of the BCR gene result in the synthesis of a protein that has the capacity to dimerise. Dimerisation transphosphorylates and then aurtophsophorylates the the kinase fully activating it. The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known but activation of  phosphatidylinositol kinase, RAS/Mitogen activated protein kinase and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways are involved in cellular growth and differentiation. The BCR-ABL1 kinase also phosphorylates proteins involved in adhesion and migration and this may have a role in premature release of myeloid cells in circulation. CML cells have a two to sixfold increase in reactive oxygen species and have impaired DNA repair. Reactive oxygen species can induce DNA double strand breaks. The results is additional mutations and these are believed to be responsible for blast crisis and acclerates phase.

Tyrosine kinase inhibitors targeting the BCR-ABL1 protein induce a remission in most patients of CML. About half the patients who have achieved sustained complete molecular response relapse on discontinuation of the tyrosine kinase inhibitors. This suggests that the stemat least some CML stem cells are not BCR-ABL1 dependent for growth. Experimental observations support this hypothesis.

Targeting the BCR-ABL1 Gene

The BCR-ABL1 gene was the first gene to be targeted by a tyrosine kinase inhibitor, imatinib. Imatinib was followed by dasatinib, nilotinib, Busotinib and Panotinob. Imatanib, Dasatinib and Nilotinib are approved to first line use. Imatinib has resulted in a 85% 8 year survival. Dasatinib and nolitinib are active in imatinib resistant CML and are now approved for first line use. Drug resistance results from mutations in BCR-ABL1 kinase. The T315I mutation or the gatekeeper mutations impaires access of TKIs to the BCR-ABL1 kinase making most drugs inactive. Panotinib can inhibit the T315I mutation.


Further Reading

Barnes DJ Melo JV. Molecular Basis of Chronic Myleoid Leukaemia. In Chronic Myeloproliferative Disorders: Cytogenetic and Molecular Anomalies. Bain Barbra J (Ed) 2003.


Dominant-Negative Phenotype

The human genome has two alleles of any gene. An allele may be dominant, recessive or co-domainant. Only one copy of an allele is needed for a dominant allele to express. A recessive allele expresses only when both the alleles are recessive.

The A and B blood group allele is dominant over the O group but are co-dominant with respect to each other. Individuals having genotype AA or AO express the A blood group antigen. Similarly individuals having genotype BB or BO express the B blood group antigen. An individual having genotype AB express both the blood group antigens. O is a recessive allele and only individuals with genotype OO have blood group O.

The biochemical basis of the ABH system is modification of the H antigen by enzymes encoded by the genes of blood group substances. Addition of N-acetylgalactosemine by the glycosyltransferase encoded by the allele encoding for A blood group converts the H antigen to A group antigen. Similarly the addition of galactose results in the synthesis of B group antigen. The allele for O group has a mutation and does not add any sugar.

Recessive genes are genes with mutations that result in loss of function. This is the case with the O blood group gene or with a thalassaemia gene. If recessive allele is present in the heterozygoyus state the product of the dominant allele, which encodes a functional protein, ensures there is synthesis of functional protein. The levels of encoded proteins in the presence of a mutant and recessive gene may be lower than in an individual without the mutant gene. Normal function is possible even with reduced levels of the encoded protein in almost all individuals and no disease occurs. This is the case with individuals with thalassaemia trait who have lower haemoglobin than normal individuals but this does not cause symptoms related to anaemia.

Some genes that encode for non-functional proteins manifest even when heterozygous with a functioning gene. These genes are known as dominant-negative. These genes usually encode for proteins that are dimers or multimers. The recessive gene has the ability to “poison” the components of the dimer/multimer produced by the normal genes and render them functionless.

Recessive genes encode for proteins that cannot form dimers/multimers. The product of these genes has no effect on the activity of the protein encoded by the normal allele. The levels of proteins may be reduced but all the protein synthesized by the dominant gene is functional. Usually the levels of proteins fall to half that in normal. This is almost always sufficient for normal function.


Figure 1. The dominant negative phenotype


The dominant-negative genes have the capacity to form dimers/multimers and also also have the capacity to inhibit the function of the product of the normal gene (figure 1). A patient carrying a dominant-negative allele has reduced levels of functional proteins because of inhibition of function by the product of dominant negative allele. If one considers a patient who has a dominant negative mutation for a protein that is a homodimer, as is the case with factor XI, 75% of the factor XI synthesized will have the monomer produces by the dominant negative gene, Only 25% of the factor XI will be functional. This is consistant with the observation that these patients have factor XI levels between 20-30%. The situations in multimeric proteins and those proteins that are hetrodimers/hetromeltimers is more complex but the principle remains the same.

Factor XII

Factor XII is a coagulation factor that initiates the intrinsic (contact) coagulation system. It is for this reason that it is also known as contact factor. It was described in 1955 in a patient John Hageman and hence it is also known as Hageman’s factor. It does not have a role in normal coagulation but may provide a link between inflammation and coagulation. It may have a role in pathological thrombosis.



The Gene for factor XII is situated on chromosome 5 (5q33-qter). The gene is 12 kilobases in length and contains 13 introns and 14 exons. It has an oestrogen responsive element in it’s promoter.


Biochemistry of Factor XII

Factor XII is a 596 amino acid single chain β-globin zymogen with a molecular weight 80kDa. It is primarily produced in the liver. Factor XII is 17% glycosylated. The plasma concentration of factor XII is 40 μg/ml (500 nmol/L) and it has a half-life of 2 to 3 days.


It is the following domains (from the N-terminal to the C terminal)

  1. N-terminal fibronectin domain type II (exons 3,4)
  2. Epidermal-growth-factor like domain (exon 5)
  3. Fibronectin domain type I (exon 6)
  4. Epidermal growth factor like domain (exon 7)
  5. Kringle domain (exon 8 and 9),
  6. Proline rich region
  7. The catalytic domain (exon 10-14) at the COOH end.

Kallikrein splits the bond between Val352 and Arg353 activating factor XII to XIIa (see activation). activation converts factor XII into a two chain structure held together by a disulfide bond between Cys340-Cys367.  The heavy chain is responsible for binding of factor XIIa to anionic surfaces (52 kDa) and a light chain (28 kDa) that has the enzymatic activity.



Factor XII can be activated to XIIa by the following

  1. Contact with negatively charged surfaces: Factor XII is activated by contact with negatively charged surfaces e.g. glass, kaolin, dextran sulphate, ellagic acid and bismuth subgallate. This rection forms the basis of the activated partial thromboplastin test. As none of these activators are encountered under physiological circumstances it is not the mechanism by which factor XII is activated in vivo for normal haemostasis. It has been hypothesized that contact with anionic surfaces provided by polyphosphates results in a conformational change and activation of factor XII. The mechanism of this activation is not clear.
  2. Activation of By Kallikrein: Factor XII, prekallikrein and high molecular weight kininogen can form a complex on anionic phospholipids of membranes. This, as discussed above, leads to a conformational change in factor XII leading to it’s activation. XIIa splits kallikrein from prekallikrein that in turn activates factor XII leading to a mutual activation loop. Activation involves a cleavage of the peptide bond between Arg 353-Val354 that converts a single chain factor XII to a two chain factor XIIa that are connected by a single disulphide bond between Cys340-Cys367.



Actions of Activated Factor XIIa

Factor XIIa activates coagulation by the intrinsic pathway by activating factor XI. The ability of factor XIIa to split prekallikrein to kallikrein links it to inflammation and fibrinolysis. Kallikrein is converts plasminogen to plasmin, pro-u-PA to u-PA and cleaves high-molecular weight kininogen to give bradykinin. Factor XII is pro-inflammatory and promotes thrombolysis by these actions.




The principle inhibitor of factor XIIa is C1-inhibitor. Antithrombin, α1-antitrypsin, α1-antiplasmin and α2-macroglobulin are the other inhibitors of factor XIIa


Role of Factor XII in Haemostasis

Patients with factor XII deficiency do not suffer bleeding following trauma or surgery despite having a prolonged aPTT. The role of factor XII in haemostasis is complex and unclear. There are many divergent pieces of evidence from experimental and epidemiological studies. These include:

  1. Deficiency in factor XII in mice has been shown to protect against thrombosis induced by injury.
  2. Anti-factor XII monoclonal antibody has been shown to reduce fibrin formation in collagen coated tubes perfused by human blood.
  3. The same antibody has been shown to reduce platelet and fibrin deposition in a baboon graft model
  4. Epidemiological studies have shown an inverse relation between factor XII levels from between all cause mortality and factor XII levels for factor XII levels between 10-100% but no increase in patients with factor levels ≤10%.

Factor XII and Disease

Mutations in factor XII have been associated with type III hereditary angioedema (HE) type III. Unlike patients of type I and II HE who have a defect of C1 inhibitor, patients of Type III HE have point mutation in factor XII resulting in substitution of threonine at position 309 with arginine or lysine.



Factor XI

Factor XI, also known as plasma thromboplastin antecedent, is a zymogen of factor XIa. It is involved in activation of the intrinsic coagulation pathway. Unlike factor XII, prekallikrein and high-molecular weight kininogen, deficiency of factor XI causes bleeding and factor XI has a physiological role in coagulation.


The gene for factor XI is located on chromosome 4 at 4q35. It is 23 kilobase long and has 14 introns and 15 exons.


Factor XI is disulphide linked dimer made of two homologous chains. Each chain is 80kDa and has 607 amino acids. 5% of the weight of factor IX is carbohydrate. The amino end has four 90-91 amino acid domains known as apple domains. These are homologous with similar domains found in prekallikrein. The carboxy end has a serine protease domains. Apple 1 domain mediates formation of complex with high-molecular weight kininogen, apple 2 domain mediated binding with factor IX, apple 3 domain binds factor IX, platelets and heparin and apple domain 4 has the dimerization site and is involved in association with factor XIIa.


Factor XI is activated by a cleavage of the Arg369-Ile370 bond. It is activated by factor XIIa, α-thrombin and factor XIa. Deficiency of factor XII does not cause bleeding but deficiency of factor XI does. The activation of factor XI by XIIa does not appear to have a physiological role. α-Thrombin is believed to the physiological activator of factor XI.


Factor XIa activates factor IX to IXa. Factor IXa is the enzymatic component of the factor X activating complex generated by intrinsic pathway. Factor XIa is a part of a positive feedback loop that activated prothrombin to thrombin via factors XI and X. The physiological role of factor XI seems to be to reinforce formation of thrombin.

Regulation of Factor XIa

Factor XIa is inhibited by plasma serpins. These include antithrombin, C1-inhibitor, protease nexin 1, and protein Z–related protease inhibitor, plasminogen activator inhibitor-1 and protein C inhibitor. Heparin enhances the inhibitory effects of srepins.

Transferrin and Transferrin Receptors

Free iron is toxic by its ability to generate oxygen free radicals and cause damage to macromolecules. Iron in transported in the plasma bound to transferrin. Uptake of transferrin by the cell is mediated by transferrin receptors of which there are two types transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2).


Transferrin (Tf) is an iron transport protein synthesised by the liver responsible for iron transport of iron. It is coded by the gene TF on 3q22.1 (OMIM 190000) There are 30 variants of Tf . Tf C is found in majority of individuals. Tf is synthesised in the liver as a single chain 80kDa in size. Each transferrin can bind two ferric atoms in a pH dependent manner. Alkaline pH promotes binding and acidic pH promotes release. Tf may exist as api-Tf, monoferric Tf and diferric Tf. Normally Monoferric Tf dominates. Diferric Tf dominates in iron overload.

Atrasnferrinaemia is characterised by hypochromic microcytic  anaemia with iron overload.

Transferrin Receptor

Two transferrin receptors have been identified TfR1 and TfR2. TfR1 is found in all cells  while TfR2 is found mainly in hepatocytes

Transferrin Receptor (TfR1) encoded by the TFRC gene on 3q29 (OMIM: 190010). It consists of two similar 760 amino acid peptide chains held together by a disulfide bone.  It binds transferrin in a pH dependent manner binding at physiological pH and releasing at acidic pH. On binding transferrin the the receptor is endocytosed. A V -type proton ATPase acidifies the endocytosed vesicles. Acidification weakens the binding of iron with transferrin releasing the iron in the vesicle.  The released ferric iron needs to be reduced to ferrous iron by  STEAP3. STEAP3 is an endosomal ferrireductase. Reduces iron is transported to the cytosol by DMT1.  TfR1 is recycled back to the surface where it is free to bind another transferrin.

Transferrin Receptor (TfR2) shares about 45% homology with TfR1. It had a lower affinity for transferrin and it’s role in transferrin uptake is not clear. It is involved in regulating hepcidin. Mutations of TfR2 are associated with haematochromatosis


Hyperleukocytosis and Leukostasis

Leukostasis is a oncological emergency seen in patients with leukaemia who present with pronounced leucocytosis. It is seen in about 5-30% of adult acute leukaemia cases. It results from slugging of microcirculation by leucocytes.  It is associated with a mortality of 20-40%.

Pathogenesis of Leukostasis

Manifestations of leukostasis result from impaired circulation in the affected vascular bed. Leukocytosis impairs circulation because of

  1. Increased viscosity
  2. Formation of intravascular leukocyte aggregates (white bland thrombi)
  3. Increased adhesion of blasts to the endothelium

Cells increase the viscosity of blood hampering flow through microcirculation. Pliability of blood cells is important for maintaining blood flow in microvasculature. The biconcave shape of the erythrocytes provides them with deformability allowing smooth passage through the microvasculature. Leukocytes are less pliable than erythrocytes.  Normally the number of leucocytes is a small fraction of the number of erythrocytes. The contribution of leucocytes to blood viscosity in minimal.

Blood viscosity increases with leucocytosis. The increase is related to the leucocyte count, size of the leukocytes and the deformability of the leucocytes. Blasts are less deformable than mature cells. Myeloblasts are larger and less deformable than lymphoblasts. Cells of the monoblastic series are the least deformable. Lymphocytes are the smallest and have the least impact on the viscosity amongst all leucocytes.

Blasts secrete cytokines like IL-1β  and TNF-β. These lead to up regulation of adhesion molecules like ICAM-1, VCAM-1 and E-selectin that increases the adhesion of blasts to the endothelium. Adhesions of blasts to microvasculature further diminish blood flow.

The vascular beds most commonly affected are the lung, CNS and the eye. Tissue hypoxia resulting from impaired circulation is believed to contribute to elevated LDH seen in acute leukaemias. 

Clinical Features

About 5-13% of acute myeloid leukaemia and 10-30% of acute lymphoblastic leukaemia have hyperleukocytosis. The symptoms of leukostasis are related to the ischaemia in the affected circulatory bed. The commonest vascular beds affected and the symptoms attributable to these beds are listed below.

Organ Manifestation
Brain Stupor
Eyes Blurring of vision
Lungs Dyspnoea
Kidney Azoaemia
Heart Arrhythmia
Penis Priapsim

Examination of the fundus shows papilledema, blurred disc margins, dilated blood vessels, and retinal haemorrhages.

The leukocyte count  at which symptoms develop depends on the type of leukaemia. Symptoms develop at the lowest counts in acute myeloid leukaemia. Patients of chronic lymphocytic leukaemia may not have symptoms of hyperleukocytosis at leucocyte counts as high as 400X109/L. Leukostasis is associated with increased morbidity and mortality.


Leukostasis is clinical diagnosis of exclusion. It should be considered in any patients with lung or CNS symptoms who has hyperleukocyosis. Hyperleukocytosis has been variably defined as a count of 50X109/L or 100X109/L. Symptoms are likely to occur at lower counts in patients with acute myeloblastic leukaemia particularly when there is a monocytoid component. Patients with chronic lymphoblastic leukaemia tolerate counts as high as 400X109/L without symptoms. The conditions that can mimic leukostasis include

  1. Pulmonary infection
  2. Pulmonary embolism
  3. Pulmonary oedema
  4. Pulmonary haemorrhage
  5. Transfusion-related acute lung injury  is blood products have been transfused
  6. CNS infections – meningitis and encephalitis
  7. Conditions causing acute mental status change

The x-ray findings include diffuse interstitial or alveolar infiltrates. It can be normal in early stages. Examination of the fundus is important.


Leukostasis is associated with a mortality of 20-40%. The treatment of leukostatsis is to rapidly reduce the leukocyte count. Three methods of rapidly reducing leucocyte counts are induction chemotherapy, leukocytopheresis or low dose chemotherapy. Each of these methods are supported by theoretical arguments. Induction chemotherapy is the definitive therapy for leukaemia. Whether leukocytopheresis or hydroxyurea add to the benefit of induction chemotherapy is not clear. The use of hydroxyurea and leukocytopheresis is dictated by the experience of the treating centre. The procedures are usually resorted to with the belief that induction therapy with a very high leucocyte count may increase the risk of tumour lysis. 

Leukocytopheresis: Leukocytopheresis is used because it rapidly brings down the leucocyte count without causing lysis of blasts. It has the theoretical advantage of reducing the risk of tumour lysis and reducing mortality. This has never been proven in clinical trials. Two procedures needed about 12-24 hour apart. Leukocytopheresis is indicated in

  1. Symptomatic patients with AML with leucocyte counts more than 50X109/L and ALL with counts more than 150X109/L.
  2. Asymptomatic patients with AML and leucocyte  counts >100 X 109/L
  3. Asymptomatic patients with ALL with leucocyte counts >300 X 109/L
  4. CML patients who are symptomatic with leucocyte counts greater than 150X109/L,
  5. CLL patients who are symptomatic with leucocyte counts greater than 500X109/L. 
  6. It should not be performed in patients with acute promyelocytic leukaemia symptomatic or asymtomatic

The procedure involves insertion of a catheter. This may be associated with an increased risk of bleeding as these patients have thrombocytopenia.

Low Dose Chemotherapy: Like leukocytopheresis the value of low dose chemotherapy has not been proven. The following interventions may be used (with leukocytopheresis)

  1. Acute myeloid leukaemia should be treated with hydroxyurea in a dose of 50-100mg/Kg. It may be administered as a single or multiple doses.
  2. Acute lymphoblastic leukaemia may be treated with steroids with or without vincristine. 

Induction chemotherapy: Induction chemotherapy is the definitive therapy for acute leukaemia. High counts are associated with a higher risk of tumour lysis resulting in an apprehensions of starting chemotherapy in patients with very high leucocyte counts.

Other measures: Cranial irradiation and dexamethasone has been used in patients with CNS symptoms. Blood transfusions can increase viscosity and may worsen symptoms of leukostasis. One needs to be conservative about red cell transfusions till the leukocyte counts become normal. Transfusions in patients with symptoms attributable to anaemia should not be held back. 




Cell catabolise proteins by two pathways, lysosome and proteasome. Proteasome is an ATP dependent pathway that plays a critical role in growth and development of the cell by degrading regulator of cell cycle. Proteasome degrade misfolded or unfolded proteins. It also breaks down peptides from infecting organisms for antigen presentation as a part of immune response. As the system is essential for life and is involved in a wide variety of critical processes, it was believed that proteasome was not a therapeutic target. However the first proteasome inhibitor, bortezomib, has shown t be a very effective therapy for myeloma patients.

The Structure of Proteasome

Proteasome has two components, the 19s cap and the 20s core.

1. 19s cap: The 19s cap has 16-18 sub-units out of which 6 have ATPase activity. The 19s cap needs energy to unfold proteins and transfer them to the core.

2. 20s core: The 20s core consists of four stacked rings two inner and two outer that enclose a chamber. Each ring is made up of seven subunits. The inner rings are made of β-units and have three proteolytic sites per ring facing into the chamber. The outer rings are made of α subunits and control the aperture of the chamber. The aperture is large enough to allow only one peptide.

Mechanism of Proteasomal destruction

Multiple molecules of ubiquitin are added to proteins marked for destruction. Ubiquitin is a highly conserved 76-aminoacis peptide. It has a terminal glycine thru which it attaches to other peptides and a lysine at position 48 to which another molecule of ubiquitin can attach via the second ubiquitin’s terminal glycine. The cap 19s unit of proteasome recognizes peptides that have at least 4 ubiquitins, deubiquitinated and unfolds such molecules passed them on to the core. Proteolysis takes place as the peptide is passing through the core producing smaller peptides that exit from the other end. These peptides are converted to amino acids by peptidases.

Ubiqutination of proteins takes place in three steps.

1. Activation of ubiquitin activating enzyme (E1) by addition of ubiquitin in an energy dependent reaction.

2. E1 transfers the ubiquitin to ubiquitin conjugating enzyme (E2)

3. The ubiquitin ligases (E3) transfer the ubiquitin from E2 forming a bond between the terminal glycine of ubiquitin to the NH2 group of lysine on the protein to be targeted for destruction.

4. The above steps are repeated an multiple molecules of ubiquitin are added to the first ubiquitin by formation of an isopeptide bond between the terminal glycine of the ubiquitin to be added and the lysine at position 48 on the ubiquitin molecule that is attached to the peptide.

The protein specificity of the ubiquitination is a result of specificity of E3 ligases for proteins. There are almost 600 ligases likely to be present. Each of these ubiquitinate a different protein(s). Protein destruction is controlled by controlling the E3 ligase.

Inhibition of Proteasome

Proteasome in view of it widespread distribution and participation in many reactions critical for cell survival was not though to be a good therapeutic target. The first proteasome inhibitor in clinical practice, bortezomib, has radically changed the outlook of multiple myeloma. It is also is used in mantle cell lymphoma.