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Future Possibilities for the Development of Treatments of
Leukemia
F. Bergel and K. R. Harrap
Cancer Res 1965;25:1643-1648.
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Future
Possibilities
for the
Development
of Treatments
of
Leukemia'
F. BERGEL AND K. R. HARRAP
Chester
Realty
Research
Institute,
Institute
of Cancer
Research:
Royal
Cancer
Hospital,
London
SUMMARY
To assess future possibilities of treatments
of leukemia, biochemical and biologic
characterizations
of the disease under discussion have been reviewed.
These char
actenizations have been used as base lines for speculations and have been summarized
innTable 1. A distinction has been made between drugs that destroy leukemic cells
and homeostatic
regulators that might achieve palliation or cure by redressing im
balances inn the leukopoietic systems.
Someone wise has said : “Itis always better to speak
after the event as an historian than before the event as a
prophet. “It is therefore a formidable task even to at
tempt crystal gazing at a time when so much work directed
toward a solution of the problem under discussion is in
progress all over the world. A good prophet is a person
who has taken in all the relevant and established facts of a
case and has digested them properly.
Then from his
computing mind may jump, like Pallas Athene out of the
head of her father Zeus, a well-armed idea or ideas, which
may prove to represent reliable forecasts for the future.
This contribution
to this symposium is based on the
authors' restricted knowledge of this field, and in specu
lating about the future developments
of leukemia treat
ments, they must stick out their necks and be prepared
to have their heads chopped off even by their kindest col
leagues.
As far as we are aware, the gulf between chronic and
acute fornis of leukemia may not be as wide or deep as
textbooks
convey.
The situation
is complex; further
more, it seems that the dividing line between the various
forms of acute leukemia—myelogenous,
lymphoid, mono
cytic—is sometimes not so sharply defined as the clinician
or hematologist may wish for his diagnostic and prognostic
considerations or for his choice of therapy.
It is outside
our competence to take sides, and an attempt is made 1st
to assess the past, present, and future status of the bio
chemical and biologic base lines of acute leukemias, ex
trapolating
where necessary from considerations
of the
chronic disorders.
In this way it may be possible to find
leads to potentially more effective therapies of this group
of diseases.
Although the causative or etiologic factors
ought to play an important part in assisting the search for
improved
methods
of treatment,
we are refraining
from
any detailed discussion on such leukemogenic studies in
the framework of this paper.
The same applies to the
physiologic role of leukocytes.
For information on these
aspects of the over-all problem, readers should consult
extensive reviews such as those of Hayhoe (30) and Bun
dette (13).
BIOCHEMICAL
The biochemist
ennces at the
BASIS
has tended to look for metabolic
molecular
level
between
leukemic
differ
and
con
respomiding normal cells, as has been done with the celLs
of other camicerous diseases, hoping to discover some
unique aspect of metabolism,
which may be exploited
therapeutically.
As yet, however, no such qualitative
differences have been identified ; only quantitative
maui
festations
have become
apparent.
A further difficulty confronting the biochennist studying
the acute leukemias is the absence of a suitable expenimen
tal system innanimals that mutates satisfactorily the situ
ationi inn man.
This makes it very difficult to study the
progressive biochemical changes taking place during leu
kemogenesis.
Owing to their insidious onset, the chronic,
and sometimes acute, leukemias are well established before
a diagnosis is made, and at this stage one may be observing
metabolic abnormalities
that are attributable
to a new
autonomous population of abnormal leukocytes.
Thus it
remains
important
to
distinguish
betweemn
metabolic
changes that are significant to the etiology of the disease
and changes that are significant to its therapeutic control.
Naturally,
one cannot in the framework of this paper
mention all the metabolic studies in this field, and con
sequently a somewhat arbitrary selection of comparative
data has been made.
1 This work has been supported
by grants to the Chester
Beatty
To begin with energy metabolism, Beck (3, 4) made a
Research Institute (Institute of Cancer Research: Royal Cancer quantitative
study of the aerobic glycolysis of circulating
Hospital) from the British Empire Cancer Campaign, the Medical
leukocytes from patients with chronic myelogenous and
Research Council, and the National Cancer Institute, NIH,
lymphatic,
but unfortunately
not with acute, leukemia.
USPHS.
1643
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Cancer Research
1644
He attributed
the low rate of aerobic glycolysis in these
cells to a deficiency in the rate-limiting
enzyme hexoki
niase. This also accounted indirectly for the increased
oxidat ion of glucose by way of the penitose shunt.
The
sanne author
(5) has measured
lactic
LEUCOCYTES
acid dehydrogenase
praisal in the light of the more recently discoveredisoenzy
matic properties of LDH.
The pattermn of isoenzyme distni
bution may be an iniportant factor in controlling leukocyte
metai)olism.
Investigationsby
Cahnn et al. (14) have shown
that there are 2 “pure―
modifications of LDH, character
istic of heart (HHHH)
arid nnuscle (M1\IM1\I) tissue,
respectively;
binonnial
conibination
of the
isoenizymes inn leu
by Goldman
and
Kaplan (26) and by Dioguardi et al. (20). The deviations
from the distribution
pattermn present in healthy leuko
cytes
are nnost niarked
iii cases
of acute
leukemia;
less
extreme variation is found inn the leukocytes of acute
leukemia iii remission amid of chrornic myeloid leukemia.
In cases of leukocytosis, the isoenzynie patterns are similar
to those inn normal leukocytes.
In continnuinig studies of
this I)henomennonn arid extending them to other isoenzyme
populations,
it may
be possible
to account
for many
of
the quantitative
differences innthe biochennistry of normal
arid leukemic leukocytes and at the same time to provide
a nnore reliable
basis for future
chemotherapy.
The presence of low levels of alkaline phosphatase in
leukocytes froni chronic myeloid leukennia or nnyeloblastic
leukennia is well known (30). Inn nornial individuals ad
mimnistratiomi
of
ACTH
or
cortisol
elicits
an
increase
in
grannulocyte alkaline phosphatase
activity (49) (cf. Ref.
39). Iii comntrast, the same drugs given to patients with
myelogenous leukemia do not stinnulate syrnthesis of this
enzyme, which indicates the possible failure of control
mechamsnns
mi this disorder.
One of the metabolic patterns that appears to be sig
nificantly altered, at least inn myelogenous leukemia, is
that of the sulfur amino acid derivatives. Thus Conto
poulas arid Andersomi (17) claimed to have detected ab
normally high levels of glutathione
in leukocytes from
patients
with
acute
myelogennous
leukemia,
and Weisber
ger aind Levimne (51) inoted that these cells appear to have
an elevated avidity for the sulfur amino acids, cystine,
cysteine, arid nnethionine, compared with normal leuko
cytes.
Following
these observations
problenn of resistance
to busulfan
and provoked
(Myleran)
by the
in patients
with chronic myelogenous
leukemia,
we studied levels of
soluble @rulfhydryl and disulfide compounds
in the blood
fractions
2 The
froni a number
following
hydrogenase;
nucleic
acid;
amide
adenine
of leukemic
abbreviatiomis
are
patients
used:
Li)H,
ACTH, adrenocorticotropiri;
RNA,
ribomiucleic
dinucleotide
amide adenine dinucleotide
ribonimcleicacid.
acid;
NAD
amid its reduced
S 32°/oSS
•
UNTREATEDCML
BUSULPHAN OR X-RAY-TREATED
710/0ss
CML APPROACHING BLAST
CRISIS
0°/aSS
CML IN REMOSION
.
.
0°/ass
40°/aSS
4 constituent
polypeptide umnitsresults in the formation of 5 independent
modifications of LDH, which cain be separated arid dis
tinguished by their different enzynnic behavior.
Abnor
malities
iii the distribution
of LDH
kennic leukocytes
have beemi recorded
ERYTHROCYTES
I 0°/aSS •
0°MSS
NORMAL CONTROLS
(LDH)2 activity at the cellular level, whereas other work
ens (53) have examined the levels of this enzyme in the
sera of leukemic patients.
All these results need reap
@
Vol.25,October1965
lactic
(29).
acid
In
de
DNA, deoxyribo
amid NADH,
nicotin
form ; NADP,
nicotin
phosphate ; and mRNA,
messenger
CHART
1.—Soluble
SH/SS
levels
in chronic
myeloid
leukemia
(CML) and in acute transformation.
summarizing
leukocytes
the results,
of all patients
Chart 1, it was found that the
with
chronic
myeloid
leukemia
contained oxidized glutathione,
the gross concentration
being dependent on the state of the disease and the course
of therapy: e.g., busulfann lowered the soluble thiol and
disulfide levels by a factor of approximately
10. No such
disulfide was detected in normal leukocytes.
In 2 patients
who had been maintained on busulfan during the chronic
phase
of myeloid
leukemia
until
they
became
resistant,
surprisingly, the presemice of oxidized glutathione in their
erythnocytes
was demonstrated
before the onset of an
acute blast-cell transformation.
This observation makes
it desirable to extend such investigation to acute leukemia
cases. Furthermore, there is an indication from this work
that the leukocyte population in well-controlled cases of
chronic myeloid leukemia, although apparently morpho
logically normal, is biochemically
still abnormal, since
soluble disulfide remains in these cells.
It is interesting to note that the coenzyme closely con
nected with sulfur metabolism—pyridoxal
phosphate—is
low in the leukocytes and plasma of patients with chronic
and acute myeloid leukemia (50). This prompted us to
examirne the conversion of pynidoxine (vitamin B6) to the
coenzyme in leukemic patients.
The results (Hanrap,
K. R., unpublished results) indicate that in patients with
chronic and acute granulocytic leukemias there is a dimin
ished
capacity
to convert
pynidoxine
into pynidoxal
phos
phate.
Although DNA and RNA metabolism is important for
the development
of a rationale for the treatment
of
acute leukemia, space does not permit a consideration in
any detail.
The topic will be treated briefly further on.
BIOLOGIC
BASIS
Suitable leads derived from the experimental
biologic
investigations
may be found among cytologic studies,
assisted by tissue culture and transplantation
technics;
genetic research into the possible prevalence of chromo
somal aberrations; and immunologic and virus studies.
It is well known that the study of leukemic and normal
leukocytes and related cells (apart from those in blood
samples directly transferred to the microscope slide) was
not a great success when they were grown in culture media
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Research.
BERGEL
AND H@rtRAP—Development
or transplanted, for instance, into the pouch of the cortison
treated hanister.
Human material proved to be intrac
table inn the latter case, and the application of various
technics of “tissueculture― with blood or bone marrow
cells (cf. Ref. 38) showed definite limitations
and did
not produce clear-cut results.
Morphologic changes fre
quently occurred during the period of successful growth.
Nevertheless,
it is suggested that further efforts inn this
field would be worth while, perhaps by using borne nnarrow
in the fornn of “organcultures― (cf. Ref. 1), checking the
nnorphology over short periods inncomparison with miormal
material, arid exposing both to old arid new drugs.
It
remains
to be seen whether
the transplantation
of hunnan
leukemic bone marrow into animals is feasible, omice a
better knowledge of imniunnologic tolerance has been
achieved.
It would certainly assist in the assessnnennt of
any novel remedies if such hetenologous bone marrow
could be grown innthe animal as a test system, thus elim
mating prelimimiary clirnical risks. The problem of the
relationship
betweemn the foreign host and the leukennic
cell will have to be resolved.
The
discovery
of chromosomal
irregularities—e.g.,
the
Philadelphia chromosome in cases of chronic gramnulocytic
leukemia—has opened up mew approaches to cytogenetic
aspects of a sniall number of diseases.
It has not yet
been established whether acute leukemias, where frequent
chromosome alteratiomis have been noted, come into this
category.
Obviously, more detailed investigations
of the
chnonnosomal DNA of leukemic cells (15) have to be
carried out during the coming years to place cytogemnetic
aberrat ions on a safer basis. This aspect has a beaning
on the proposals to be made below, where the possibility of
the effects of extraneous niucleic acids will be discussed.
This brings us to the role of immunology in the field of
leukemias and, arising out of it, the possible treatment
by antisera.
Specific surface antigens have been dis
covered inn normal leukocytes, and antisera against the
latter and a variety of leukemic cells have been prepared;
such sera show at least some in vitro effects (30). The
existence
of specifically
leukemic
antigens
of leukocytes
is
uncertain ; their presence on absence is still to be proved.
The possibility of loss of antigens during leukemogenesis
has to be considered
of that
prevailing
as well.
The situation
in other neoplastic
is reminiscent
diseases.
It seems
TABLE 1
GroupSubstancesI.“Killer―
drugsImprovements
of existing drugs and elim
ination of their resistance
Discovery of novel types, either organic
molecules or antisera, vaccines
Hormones (steroids, adrenocorticotropin,
others)II.HomeostaticCatabolic
and
zymes, metal activators
Transforming factors, nucleic acids, miucleo
proteins, polyelectrolytes
Differentiation inductors, maturation
agents
1645
Treatment
advisable to pursue this immunologic aspect to ascertain
whether there is any foundation for claims by workers
such as Dc Carvalho (19) for specific hyperinimune gamma
globulins inn animal serum (for instance, the horse amid
domikey received a preparation
from leukemic spleemis
purified by precipitationn of the “miornnal
antigens― with
antibodies against normal tissues).
It is not improbable
that refined technnics and the application
of procedures
enhancing
anntigemnicity may lead to more
In comntrast to certain
filtrable
tangible
amiimal leukemias
agents (24, 25, 28), the virus
mi animals
with
cell-free
results.
produced
or subcellular
etiology of human types has riot been proved.
experiments
material
by
particle
Imiductiomi
from
humamn
leukemic patients have sometimes shown positive results,
but the nature of the causative agent or the exact type of
the disease, for instamnce in mice, is far from clear. For
many years Dmochowski
arid his school have dennoni
strated the presence of virus-like material in samples of
malignant
tissues ; recently a claim has been made by
Melnnick et al. (34) for the presence of mixovirus-like iar
tides innthe plasma of 80 % of children with acute leuke
mia. Evidence that these particles are the cause of the
disease is still missing, especially since these “viruses―
have also beemi observed innchildren with imnfectious rinomio
nucleosis.
Consequently,
it is too early to speculate on
the possibility of producing vaccines.
FUTURE
TREATMENTS
Imi this section the emphasis is on speculation rather
than on logical deduction from established facts.
We propose that improvements
of existing treatments
(whetherthesearedesignedto improveselectivityor to
eliminate drug resistance) arid any novel forms of therapy
should be treated under the headings of (a) Group
@:
“killerdrugs― arid (b) Group II : homeostatic regulators
(restitutive agents) (6, 21). Group I aims at the destruc
tionn or total elimination of a pathologic cell type or types,
which gives the formation site of normal cells (nornnal inn
proliferation
arid maturation)
a chance to recover suffi
ciently so that only healthy leukocytes are produced at a
controlled rate.
Group I also includes antisera (as far as
they exist or will be discovered) arid hormonal substances,
though only inn pant , since the action niechannism of the
latter could be regulatory.
Group H exists now to a
lange extent as a concept ; it. includes those compounds,
materials, amid extracts, nnaimnly of natural origin, which
may be capable of healing lesions of a biochemical or bio
logic character, which are fundamentally
connected with
the etiology of the blood disorders under discussiomn. Such
remedies could achieve their curative effects by altering
the proliferatiomn rate of leukopoietic
systems and by
transformimig the cell types arising therefrom into niione
mature, niornial forms.
Although the practical outcome
is uncertain,
and anabolic : functiomial pro
enzymomimetic
systems,
coen
regulatorsteins,
of Leukemia
there
is evidence
for the existence
of ann ex
penimemital basis (chemistry, biochemistry, and biology of
large nnolecules and their role inncellular events (see Bergel
[7]) (Table
1).
In Group I, one could deal first with improvements
of
existinng drug types, such as anti-metabolites,
alkylating
agemits, and, in pant, hormonal substances.
The eliminna
tion of drug resistance is closely connected with the prob
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Research.
1646
Cancer Research
lems of achieving greater selectivity of aniti-leukemic ac
tion. In all this work greater attention should be paid to
biochennical abmnormalities of leukemic tissue amid cells,
arid more intemnse exploitation of even present-day knowl
edge of metabolic lesions could be attempted
(see “Bio
chennical Basis―). With the assumption that past and
curremit research by many laboratories
inn the field of
punimnearid pynimidimie amialogs, of anti-folics, and of alkyl
atinig agents is generally kmiowmn(see Ref. 16), it should
be pointed out that further innpnovements will originate
more readily from ant increased exploration of the action
mechanism of some of these drugs than from random syn
thetic programs.
Considerable progress has been made
in this respect with 6-mercaptopurine
and its derivatives
arid related
catabolic
inhibitors
(2, 22, 33, 36, 44), with
thioguamnimie (32), with azaunidine (39), with anti-folics
(10, 31, 52), amid others amid, although riot applicable to
acute leukemia, with busulfan (29, 41). However, is it
really sufficient to attack the obstacles to the control of
acute leukemia with ingenious modifications, such as the
bis(thioimiosinne)-5'
, 5―-phosphat.e
(36)
and
nicotinamide
6-niercaptopunine
dinucleotide
(2)? Of course other
changes will be rung, arid variants of anti-folics will be
niade, such as tetrahydrohomofolate
(27), which might
circurinvent amethopterin
resistance.
Yet doubts linger
in omie's nnind about the final clinical success. Perhaps
novel organic niolecules will suddenly appear (45), our
friends the senologists will produce purified hyperimmune
sera (19), or if the “hunches―
about virus leukemogenesis
(34)
solidify
preventive
imito proofs,
they
will provide
vaccines
for
treatnnennt.
However, it is felt. that more attractive
possibilities
may
exist with hormonal
substances
of the steroid and ACTH
types.
If their action and resistance mechanism could
be fully elucidated and structures synthesized that would
carry,
on the
basis
of these
elucidations,
more
specific
therapeutic features, then considerable advances could be
predicted.
A iortiomn of this horniornal activity must be due to the
influence of the compounds on homeostatic
regulation.
It is riot known whether this happens via a favorable
change of cell-membrane penetration, allowing for an im
proved t rade betweemi the inside and the outside of the
cell, or whether enzymic patterns are altered, perhaps be
cause
of the effect
of the steroids
themselves
or other
in
tercellular
constituemits on the allostenic properties
of
functional proteins (35). Whatever the nature of the
receptor with which they interact, we are convinced that
studies of possible nneans to obtaimi homeostatic regulation,
which ainis, as mentioned before, at the complete on at
least
Partial
restoration
of normality,
should
be carried
out without delay.
In our opinniomnthis could be done in many ways,
whereby the simultaneous application of a mnumbenof pro
cedunes may be necessary.
Apart from the hormonal
studies suggested above, the following may represent
working hypotheses for future treatments:
changes in the
levels on character of functional proteins by various means;
restitutioni of cofactors arid metal activator deficiencies;
alterations of genetic controls, directed riot only toward
operative but also toward regulatory ornes; and introduc
Vol.25,October1965
tion to the diseased tissues of maturation factors or neclif
ferenntiation agents.
The imbalance of biocatalysts
in leukemias could be
connected directly by extraneous application of purified
enzymes, preferably of enzymomimetic
systems (which
are much less efficacious than holoenzymes but do not
carry
immunologic
hazards)
be connected indirectly
nisms
with
and of coenzymes.
by utilization
substrates,
products,
It could
of feedback
mecha
or compounds,
which
according to Monod et al. (35), would influence the con
formational properties or tertiary and quatennany struc
tunes of endogenous
enzymes,
including
isoenzymes.
Manipulation
of the inducer-repressor
controls, if they
should prove accessible to extraneous methods, could also
be used.
The adnninistnation of exogenous enzymes on enzymo
mimetics, which catalyze degrading processes, could de
pnive the diseased sites of leukopoiesis or the circulating
abnormal cells of metabolites on which they depend more
than their healthy counterparts.
If such enzymatically
active substances
promote cellular syntheses that are
found to be suppressed on missing, the use of exogenous
enzymes could repair such deficiencies.
Examples of the
catabolic type, though not yet fully and convincingly
demonstrated
(6), are purified xanthine oxidase, crystal
line nibonuclease, a model cysteine desulfhydrase,
and
aspanaginase.
In the case of the model cysteine desulfhydrase,
con
sisting
of pynidoxal
phosphate
and
vanadyl
salts
(pyn
vanal), Bengel et al. (8, 9) have shown that it degrades
cysteine in vitro and, in collaboration with their clinical
colleagues, that a mixture of pynidoxine and sodium
vanadate can cause a fall in the total leukocyte count in
acute myeloid leukemia.
In view of the disturbance of
the pynidoxine-pyridoxal
phosphate biosynthesis
in leu
kemic patients (see above), it seems desirable to study
further the effects of a complex of vanadium and the pne
formed coenzyme or of an appropriate
cell-penetrating
derivative.
Such investigations
could be extended to
other enzymomimetic
systems, whether known now on
still to be discovered.
Whenever a malignant tissue proved to be asparagine
dependent, Broome (12) had succeeded in controlling, for
instance,
experimental
lymphomas
by the administration
of purified asparaginase
from guinea pig serum.
This
observation should also be given more attention, especially
if the diseased bone marrow of patients with acute leuke
mias depends on asparagine for its proliferative activities.
Whether catabolic or anabolic processes are involved,
it may prove easier to introduce the appropriate
coen
zymes,
especially
where
the deficiency
is not attributable
to the apoenzymes.
Thus, the low levels of NAD/NADH
in many growing tissues (37) could be replenished by ad
ministration
of lipid-soluble derivatives of NAD, which
have an increased chance to enter the target celLs. Unfor
tunately, whenever the levels of cofactors are below nor
mal, the biosynthetic processes leading to them are defi
cient as well, and it is consequently no use to offer the
organism more nicotinamide
or, as mentioned
before,
pyridoxine.
A wide field of work with synthetics presents
itself to the medicinal chemist who wishes to follow the
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Research.
BERGEL
AND H@u@RAP—Development
footsteps of Montgomery et at. (36) with mencaptopunine
nucleotides on Atkinson et al. with NAD analogs (2).
Should the observation by Hannap and Speed (29) con
cerning the presence of oxidized glutathione
in blood
fractions be confirmed in acute leukemias, the use of
NADPH
(a cofactor of glutathione
reductase) could be
explored.
Assuming that the appearance
of altered isoenzyme
patterns of LDH and possibly other enzymes has any
beaning on the etiology of acute leukemia, changing them
back to normal would not be easy unless their site of pro
duction is treated.
This could be achieved only by inter
ference with the control mechanism, operative or regula
tony. Cases of altering protein biosynthesis by extraneous
means in mammalian
cells are as yet rare. Some (48)
have claimed that human culture cells (1 in 1O.@)were
transformed
from a deficient type to an enzymatically
complete type with the DNA of the latter.
Others (43)
have reported that levels of interferon in virus-infected
cultures were increased by the addition of “foreignRNA,―
on even by polyadenylic acid. Taking into consideration
the infectivity of some isolated virus RNA's and the claim
of a number of authors (see Ref. 7) of having affected
tumonigenesis and carcinolysis by administration
of flu
cleic acids, either DNA on mRNA, one wonders whether
one of the most importamnt future developments
for the
treatment of acute leukemia will be found in this direction.
The question may sound absurd at this moment : could
not some of the favorable effects of blood transfusions
(18) be caused in part by the presence of such polynucleo
tide or oligonucleotide fractions innthe cells suspended in
the transfused fluid? Having been asked specifically to do
some crystal-gazing,
however, we venture to recommend
the studies of material from such fluids, then of purified
substances, and finally of simpler polyelectrolyte
models.
We propose the assessment of possible favorable effects
of bases or polybases on the cell permeability
of poly
acids, both natural
and synthetic
(47). We suggest
that strictly controlled bone marrow destruction leading
to near aplasia (a side effect of radiotherapy
on chemo
therapy on immune suppression, which clinicians try to
counteract
with
autologous
bone
marrow
or enythnocyte,
of Leukemia
1647
Treatment
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1. Ambrose, E. J., Yarnell, M. M., Easty, D. M., Shepley, K.,
Tchao, R., Easty, G. C., O'Connor, T., and Wylie, J. A. H.
Tissue and Organ Culture Studies. B.E.C.C. Ann. Rept., 41:
122,1963.
2. Atkinson, M. R., Jackson, J. F., Morton, R. K., and Murray,
A. W. Nicotinamide 6-Mercaptopurine Dinucleotide and Re
lated
Compounds—Potential
Sources
of 6-Mercaptopurine
Nucleotide in Chemotherapy. Nature, 196: 35-36, 1962.
3. Beck, W. S. The Control of Leukocyte Glycolysis. J. Biol.
Chem., 232:251—70,
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