7 Therapeutic Resistance in Leukemia

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137

From: Cancer Drug Discovery and Development: Cancer Drug Resistance Edited by: B. Teicher © Humana Press Inc., Totowa, NJ

Therapeutic Resistance in Leukemia

William R. Waud, ^{P h D}

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At Southern Research Institute, a series of in vivo drug-resistant murine P388 leu- kemias were developed for use in the evaluation of crossresistance and collateral sen- sitivity. These in vivo models have been used for the evaluation of new compounds of potential clinical interest. Crossresistance data coupled with knowledge of the mecha- nisms of resistance operative in the drug-resistant leukemias may identify useful guides for patient selection for clinical trials of new antitumor drugs and noncrossresistant drug combinations.

Key Words: P388 leukemia; drug-resistant tumor lines; drug resistance; therapeutic resistance.

1. INTRODUCTION

There have been major advances in the treatment of human leukemia (1–4). Patients with acute lymphoblastic leukemia afford the best example. In children, the event-free survival rate for the standard-risk patient is approximately 80% at 4 yr and approx 65%

for the high-risk patient. In adults, complete remissions occur in approximately 65–85%

of the patients; however, the majority of these patients subsequently relapse, and overall, only 20–30% are cured. For acute myelogenous leukemia, less dramatic results have been achieved. In children, the complete remission rate is approximately 85%, but only 30–

50% of the patients are long-term survivors. In adults, the majority of patients relapses, and ultimately dies from the consequences of resistant disease. Unlike acute leukemias, chronic leukemias are usually refractory to treatment. In adults, survival rates are 60–

70% and 30–40% for 5 and 10 yr, respectively.

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Despite the successes in the management of human leukemias, certain obstacles re- main. Among the most important is the development of resistance to the agents used for treatment. Understanding the resistance to various agents will facilitate the development of strategies to overcome the resistance.

At Southern Research Institute, a series of in vivo drug-resistant murine P388 leuke- mias has been developed for use in the evaluation of crossresistance and collateral sen- sitivity. These in vivo models have been used for the evaluation of new drugs of potential clinical interest. Crossresistance data, coupled with knowledge of the mechanisms of resistance operative in the drug-resistant leukemias, may yield insights into the mecha- nisms of action of the agents being tested. Similarly, crossresistance data, coupled with the mechanisms of action of various agents, may yield insights into the mechanisms of resistance operative in the drug-resistant leukemias. Furthermore, crossresistance data may identify potentially useful guides for patient selection for clinical trials of new antitumor drugs and possible noncrossresistant drug combinations.

Schabel and coworkers have published the most extensive summary of in vivo drug resistance and crossresistance data available (5). Their initial report included results of in vivo crossresistance studies of 74 antitumor drugs in 12 drug-resistant P388 leukemias.

Previously, we expanded this crossresistance database for the drug-resistant P388 leuke- mias to include more clinically useful drugs, and we updated the database to include new candidate antitumor agents entering clinical trials (6). In this chapter, we have expanded this crossresistance database for the drug-resistant P388 leukemias to include four addi- tional drug-resistant leukemias and have focused on clinically useful drugs. Furthermore, we have used the crossresistance data to gain insights into the mechanisms of resistance operative in these drug-resistant leukemias.

2. DRUG-RESISTANT P388 LEUKEMIAS 2.1. Selection Procedures

Eleven of the 16 drug-resistant P388 leukemias were developed at Southern Research Institute. B6D2F

mice (CD2F

mice for N,N'-bis(2-chloroethyl)-N-nitrosourea [BCNU], melphalan [L-PAM], etoposide [VP-16], and paclitaxel [PTX]) bearing intraperitoneal (ip) implants of 10

P388/0 cells were treated intraperitoneally with the drug of interest.

When half the mice had died, tumor cells were harvested from a survivor that showed frank accumulation of ascites fluid. Transplantation of 10

cells intraperitoneally to healthy mice and ip treatment with the drug of interest was repeated until there was no further increase in resistance to the drug of interest.

The dosages and treatment schedules for the eleven drugs were as follows:

1. Actinomycin D (ACT-D): first treatment, 0.05 mg/kg/dose, days 4–12 postimplant; sec- ond treatment, 0.5 mg/kg, day 5; third treatment, 0.3 mg/kg, day 7; and subsequent treatments, 0.3 mg/kg, day 4.

2. 1- β-

-Arabinofuranosylcytosine (ara-C): 20 mg/kg/dose, days 1–9.

3. BCNU: first treatment, 20 mg/kg, day 3; and subsequent treatments, 25 mg/kg, day 2.

4. Cyclophosphamide (CPA): first treatment, 25 mg/kg/dose, days 1–24; second to fourth treatments, 25 mg/kg/dose, days 1–9; and subsequent treatments, 100 mg/kg, day 4.

5. Doxorubicin (ADR): first treatment, 1.5 mg/kg/dose, days 4–12; second treatment, 12.5 mg/kg, day 5; third treatment, 6 mg/kg, day 7; and subsequent treatments, 6 mg/kg, day 4.

6. 5-Fluorouracil (5-FU): 20 mg/kg/dose, days 1–9.

7. L-PAM: 2 mg/kg/dose, days 1–5.

8. Methotrexate (MTX): first and second treatments, 1.6 mg/kg/dose, days 1–9; third treat- ment, 2.4 mg/kg/dose, days 1–9; fourth to sixth treatments, 1.6 mg/kg/dose, days 1–9;

and subsequent treatments, 0.75 mg/kg/dose, subcutaneously, days 1–9.

9. PTX: 15 mg/kg/dose, days 1–5.

10. Vincristine (VCR): 1 mg/kg/dose, days 1, 5, and 9.

11. VP-16: first and second treatments, 40 mg/kg/dose, days 3, 7, and 11; third treatment, 40 mg/kg/dose, days 3 and 7; fourth treatment, 40 mg/kg, day 3; fifth treatment, 40 mg/kg/dose, days 3 and 7; and sixth treatment, 40 mg/kg/dose, days 3, 7, and 11.

P388/amsacrine (AMSA), P388/dihydroxyanthracenedione (DIOHA), and P388/

camptothecin (CPT) were obtained from Dr. Randall Johnson (GlaxoSmithKline, King of Prussia, PA), P388/cisplatin (DDPt) was received from Dr. Joseph Burchenal (Memo- rial Sloan-Kettering Cancer Center, New York, NY), and P388/mitomycin C (MMC) was obtained from Dr. William Rose (Bristol-Myers Squibb, Wallingford, CT). P388/0 was obtained from the Developmental Therapeutics Program Tumor Repository, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick, Maryland. P388/

AMSA was developed by serial ip passage of P388/0 cells in CD2F

mice that were treated intraperitoneally with AMSA at a dosage of 4 mg/kg/dose on days 4–10 postimplant. After nine transplant generations, the treatment schedule was changed to days 1–7 (7). P388/DIOHA was developed by serial ip passage of P388/0 cells in CD2F

mice that were treated intraperitoneally with DIOHA (NSC 299195) at a dosage of 10 mg/

kg on day 2. After 40 transplant generations, treatment was changed to 0.5 mg/kg/dose on days 1–7 (8). P388/CPT was developed by serial ip passage of P388/0 cells in B6D2F

mice that were treated intraperitoneally with CPT at a dosage of 3 mg/kg on day 2 for four transplant generations, 3 mg/kg on day 1 for the next three transplant generations, 6 mg/kg on day 1 for the next 29 transplant generations, and 9 mg/kg on day 1 for subsequent transplant generations (9). P388/DDPt was developed by serial ip passage of P388/0 cells in B6D2F

mice that were treated with DDPt at a dosage of 8 mg/kg on day 1 over successive generations until no increase in survival time was seen with any tolerated dose of DDPt (10). P388/MMC was developed by serial ip passage of P388/0 cells in CD2F

mice that were treated intraperitoneally with MMC at a dosage of 0.8 mg/kg/dose on days 1–4 over successive generations until no increase in survival was seen at an optimal dose of MMC (11).

Six of the drug-resistant P388 lines are passaged in the presence of drug. The treat- ments are as follows:

1. Actinomycin D: 0.2 mg/kg, intraperitoneally, day 4.

2. CPA: 100 mg/kg, intraperitoneally, day 1.

3. ADR: 4 mg/kg, intraperitoneally, day 4.

4. 5-FU: 15 mg/kg/dose, subcutaneously, days 1, 3, and 5.

5. L-PAM: 2.5 mg/kg, intraperitoneally, day 1.

6. MTX: 0.75 mg/kg/dose, subcutaneously, days 1–6.

2.2. Mechanisms of Resistance

Increased NAD-dependent aldehyde dehydrogenase activity has been demonstrated

to be a mechanism of in vivo resistance to CPA in both P388 (12) and L1210 (13)

leukemia.

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There has been only one report concerning the mechanism(s) of resistance for P388/

L-PAM. Harrison et al. (14) reported an elevated intracellular glutathione (GSH) concen- tration for our P388/L-PAM in comparison to P388/0. Studies conducted with L1210/L- PAM have shown the cells to have defective drug transport and increased intracellular levels of GSH and GSH disulfide (15,16).

Resistance to DDPt in P388/DDPt leukemia (selected for resistance in vivo before chronic in vitro exposure to DDPt) is multifactorial (17,18). P388/DDPt cells exhibit decreased drug accumulation, elevated GSH, and increased DNA polymerase- β activity.

We have found that our in vivo P388/DDPt contains elevated DNA topoisomerase II activity in comparison to P388/0 (unpublished results).

Whereas there appears to have been no published reports concerning the mechanisms(s) of BCNU resistance in P388 leukemia, studies have shown that in vivo L1210/BCNU, which has an elevated O

-alkylguanine-DNA alkyltransferase content in comparison to the parental L1210/0, exhibits a faster rate of repair of BCNU-induced damage to DNA (19). Furthermore, L1210/BCNU exhibits an increased activation of DNA polymerase- β ( 20).

There has been only one report concerning the mechanism(s) of resistance for P388/

MMC. Kobayashi et al. reported that the accumulation of MMC in P388/MMC cells was lower than that in the parental P388 cells (21).

For P388/MTX, there have been reports of elevated dihydrofolate reductase activity in cells selected for resistance in vitro (22) and increased expression of the mdr1 gene with little change in the energy-dependent drug efflux pump (as determined by rhodamine efflux) in cells selected for resistance in vivo (23). Even though the mecha- nisms of MTX resistance operative in P388/MTX have not been investigated exten- sively, studies have been conducted in both animal and human cells, especially with other murine leukemias (e.g., L1210 and L5178Y). Resistance to MTX has been attrib- uted to altered drug transport (24), altered dihydrofolate reductase (25), increased level of dihydrofolate reductase resulting from gene amplification (26), and defective me- tabolism to polyglutamate species (27).

Resistance to 5-FU in P388/5-FU has been attributed to reduced initial uptake of drug, decreased levels of uridine kinase and uracil phosphoribosyltransferase, and reduced levels of 5-FU nucleotides (28,29).

For P388/ARA-C, there appear to have been no published data concerning the mechanisms(s) of resistance. Studies conducted with human and other murine (L1210, L5178Y, and P815) leukemia cells resistant to ara-C have revealed several mechanisms of resistance: decreased membrane nucleoside-binding sites, decreased deoxycytidine kinase activity, increased cytidine deaminase activity, and increased intracellular cyti- dine triphosphate and deoxycytidine triphosphate pools (30,31).

For P388/ACT-D (or L1210/ACT-D), there appear to have been no published reports concerning mechanisms of resistance. Resistance to actinomycin D in Chinese hamster cells has been attributed to differences in cell membrane in comparison to parental cells, resulting in decreased drug permeability (32).

Resistance to ADR in P388/ADR leukemia (selected for resistance in vivo) is multi-

factorial (33,34). P388/ADR cells exhibit decreased drug accumulation, decreased for-

mation of DNA single- and double-strand breaks, increased GSH transferase activity,

earlier onset of DNA repair, reduced DNA topoisomerase II activity and protein levels

(due to a fusion of the genes for topoisomerase II alpha and the retinoic acid receptor

[35]), and elevated P-glycoprotein.

Resistance to AMSA in P388/AMSA (selected for resistance in vivo) has been attrib- uted in part to a decrease in DNA topoisomerase II activity (36) due to a rearrangement of the DNA topoisomerase II gene (37) and does not appear to be due to alterations in the cellular uptake or efflux of AMSA (38). Studies in our laboratories have shown that P388/

AMSA does not overexpress the mdr1 gene in comparison to the parental P388/0 (unpub- lished results).

There have been no reported studies on the mechanisms of resistance of P388/DIOHA.

Studies in our laboratories have shown that P388/DIOHA does not overexpress the mdr1 gene in comparison to the parental P388/0 (unpublished results). Mitoxantrone-resistant HL-60 cells (selected for resistance in vitro) exhibit multidrug resistance but do not have altered drug transport or overexpression of P-glycoprotein (39); however, P388 cells selected for resistance to mitoxantrone in vivo exhibit overexpression of P-glycoprotein and reduced DNA topoisomerase II activity and protein levels (40).

Concerning the mechanisms of resistance of P388/VP-16, Higashigawa et al. reported that the pyrimidine triphosphate pools were significantly decreased in comparison to the parental P388/0 line (41). Studies in our laboratories have shown that P388/VP-16 does not overexpress the mdr1 gene but does exhibit decreased DNA topoisomerase II activity in comparison to the parental line (42).

Resistance to CPT in P388/CPT (selected for resistance in vivo) has been attributed to a decrease in DNA topoisomerase I activity due to a rearrangement of the topoisomerase I gene (37). Concomitant with this change has been an increase in DNA topoisomerase II activity.

P388/VCR (chronic selection in vivo) exhibits reduced intracellular accumulation and enhanced efflux of VCR in comparison to parental P388/0 (43). The selection procedure apparently affects the observed crossresistance profile. P388/VCR (Japanese Foundation for Cancer Research), selected at 0.25 mg/kg/dose on days 2–10, is crossresistant to ADR and mitoxantrone, whereas our P388/VCR, selected at 1 mg/kg/dose on days 1, 5, and 9, is not crossresistant to ADR or mitoxantrone (Table 1) (44). Studies in our laboratories have shown that our P388/VCR does not overexpress the mdr1 gene in comparison to the parental P388/0 (unpublished results). HL-60/VCR (selected for resistant in vitro) exhib- its reduced intracellular accumulation of VCR and increased levels of three surface glycoproteins, two of which are highly reactive with a monoclonal antibody against P- glycoprotein (45). Various K562/VCR in vitro clones have been shown to have reduced intracellular accumulation and enhanced efflux of VCR; one clone was found to possess diminished amounts of β-tubulin ( 46).

Even though the mechanisms of PTX resistance operative in P388/PTX have not been investigated, studies conducted with other tumor cell lines have revealed the resistance to be multifactorial—overexpression of the mdr1 gene, molecular changes in the target molecule ( β-tubulin), changes in apoptotic regulatory and mitosis checkpoint proteins, and more recently changes in lipid composition and potentially the overexpression of interleukin 6 (47).

2.3. Experimental Design

CD2F

mice were implanted intraperitoneally with 10

cells of either P388/0 or a drug-

resistant P388. Tumor implantation day was designated day 0. Drugs were administered

intraperitoneally according to the schedules listed in the tables. Each drug was evaluated

at several dosage levels (ranging from toxic to nontoxic), with each dosage level admin-

istered to 6–10 mice. Tumor-bearing control mice (12–20 per experiment) were untreated.

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Mice were observed for lifespan. In each experiment, tumored groups were treated with a range of dosages of the appropriate drug to confirm the resistance of a drug-resistant P388 leukemia. Moreover, a drug-resistant P388 leukemia was compared directly in each experiment to P388/0, and the parallel groups of mice were treated identically with a single drug preparation. Experiments were typically repeated for confirmation.

Antitumor activity was assessed on the basis of percent median increase in lifespan (%

ILS) and net log

cell kill. Calculations of net log

cell kill were made from the tumor doubling time that was determined from an internal tumor titration consisting of implants from serial 10-fold dilutions (48). Long-term (45 to 60 d) survivors were excluded from calculations of % ILS and tumor cell kill. To assess tumor cell kill at the end of treatment, the survival time difference between treated and control groups was adjusted to account for regrowth of tumor cell populations that may occur between individual treatments (49). The net log

cell kill was calculated as follows:

Net log

cell kill = [(T – C) – (duration of treatment in days)] / 3.32 × T

where (T – C) is the difference in the median day of death between the treated (T) and control (C) groups, 3.32 is the number of doublings required for a population to increase

Table 1

Crossresistance of P388 Sublines Resistant to Various DNA and Tubulin Binders to Clinically Useful Agents

CD2F1 mice were implanted ip with 10⁶ P388/0 or drug-resistant P388 cells on day 0. Data presented are for ip drug treatment at an optimal (ⱕLD10) dosage.

Resistance/crossresistance, +; marginal crossresistance, ±; no crossresistance, –; and collateral sensitivity, =.

aTreatment schedule (Rx): A, day 1; B, days 1, 5, 9; C, days 1–5; D, days 1–9; E, days 1, 4, 7, 10.

bData from Mol Pharmacol 1990; 38:471–480.

cTreatment schedule was days 1, 5, 9.

dTreatment schedule was days 1–5.

eTreatment schedule was days 1 and 5.

1 – log

U, and T

is the mean tumor doubling time (days) calculated from a log-linear least-squares fit of the implant sizes and the median days of death of the titration groups.

Crossresistance was defined as decreased sensitivity (by >2 – log

U of cell kill) of a drug-resistant P388 leukemia to a drug compared to that observed concurrently in P388/

0 leukemia. Similarly, marginal crossresistance was defined as a decrease in sensitivity of approximately 2 – log

U. Collateral sensitivity was defined as increased sensitivity (by >2 – log

U of cell kill) of a drug-resistant P388 leukemia to a drug over that observed concurrently in P388/0 leukemia.

3. CROSSRESISTANCE PROFILES 3.1. Resistance to Alkylating Agents

The crossresistance profile of P388/CPA to 14 different clinical agents is shown in Table 2. The P388/CPA line was crossresistant to one (MMC) of the five alkylating agents, no antimetabolites, no DNA-binding agents, and no tubulin-binding agents.

Crossresistance of P388/CPA has also been observed for two other alkylating agents (chlorambucil and ifosfamide) (50). Interestingly, there are differences among these

Crossresistance of P388 Sublines Resistant to Various Alkylating Agents and Antimetabolites to Clinically Useful Agents

CD2F1 mice were implanted ip with 10⁶ P388/0 or drug-resistant P388 cells on day 0. Data presented are for ip drug treatment at an optimal (ⱕLD10) dosage.

Resistance/crossresistance, +; marginal crossresistance, ±; no crossresistance, –; and collateral sensitivity, =.

aTreatment schedule (Rx): A, day 1; B, days 1, 5, 9; C, days 1–5; D, days 1–9; E, days 1, 4, 7, 10.

bData from InVivo 1987; 1:47–52.

cTreatment schedule was day 1.

dTreatment schedule was days 1 and 5.

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three agents. Chlorambucil and ifosfamide, like CPA, each have two chloroethylating moieties, whereas MMC is from a different chemical class. Ifosfamide, CPA, and MMC require metabolic activation, and chlorambucil does not. Although P388/CPA is crossresistant to two chloroethylating agents, the line is not crossresistant to other chloroethylating agents (L-PAM and BCNU). Therefore, P388/CPA appears to be crossresistant only to a select group of alkylating agents with differing characteristics.

P388/CPA appears to be collaterally sensitive to fludarabine.

The effect of 15 different clinical agents on P388/L-PAM is shown in Table 2. The P388/L-PAM line was crossresistant to approximately one half of the agents—two of four alkylating agents, one of four antimetabolites, three of five DNA-binding agents, and one of two tubulin-binding agents. The alkylating agents involved in crossresistance represent different chemical classes. Similarly, the DNA-interacting agents involved in crossre-sistance include agents with different mechanisms of action—inhibitors of DNA topoisomerase II (AMSA and mitoxantrone) and a DNA-binding agent (actinomycin D).

However, the L-PAM-resistant line did not exhibit crossresistance to other inhibitors of DNA topoisomerase II (e.g., ADR and VP-16) or another DNA-binding agent (e.g., ADR).

The sensitivity of P388/DDPt to 17 different clinical agents is shown in Table 2. The P388/DDPt line was not crossresistant to any of these agents. Interestingly, the DDPt- resistant line was collaterally sensitive to three agents (fludarabine, AMSA, and mitoxantrone). Of these three agents, the latter two have been reported to interact with DNA topoisomerase II (51,52).

The crossresistance data for P388/BCNU have been limited to the evaluation of alky- lating agents. The crossresistance profile of P388/BCNU to four different clinical agents is shown in Table 2. The BCNU-resistant line was not crossresistant to L-PAM, CPA, MMC, or DDPt.

The crossresistance profile of P388/MMC to 13 different clinical agents is shown in Table 2 (11). The P388/MMC line was crossresistant to approximately one half of the agents—one of three alkylating agents, zero of four antimetabolites, three of four DNA- binding agents, and two of two tubulin-binding agents. The pattern was similar to that observed for P388/L-PAM.

3.2. Resistance to Antimetabolites

The effect of 14 different clinical agents on P388/MTX is shown in Table 2. The P388/

MTX line was not crossresistant to any of these agents.

The crossresistance data for P388/5-FU have been limited to antimetabolites. The sensitivity of the P388/5-FU line to three different agents is shown in Table 1. The P388/

5-FU line was not crossresistant to palmO-ara-C (a slow-releasing form of ara-C) or fludarabine (possible collateral sensitivity). Crossresistance was observed for MTX.

The crossresistance profile of P388/ARA-C to 16 different clinical agents is shown in Table 2. The P388/ARA-C line was crossresistant to members of several functionally different classes of antitumor agents—four of five alkylating agents, three of five anti- metabolites, none of four DNA-binding agents, and one of two tubulin-binding agents.

Interestingly, the line was collaterally sensitive to 5-FU.

3.3. Resistance to DNA- and Tubulin-Binding Agents

The effect of 17 different clinical agents on P388/ACT-D is shown in Table 2. P388/

ACT-D was not crossresistant to any alkylating agents or antimetabolites. It was, how-

ever, crossresistant to all of the drugs tested that are involved in multidrug resistance except for AMSA.

The crossresistance profile of P388/ADR to 21 different clinical agents is shown in Table 2. The P388/ADR line was not crossresistant to any of the antimetabolites and was marginally crossresistant to only one alkylating agent (MMC). Resistance was observed for all of the drugs tested that are reported to be involved in multidrug resistance (acti- nomycin D, ADR, VP-16, AMSA, mitoxantrone, vinblastine, VCR, and PTX). P388/

ADR was collaterally sensitive to fludarabine.

The sensitivity of P388/AMSA to 14 different clinical agents is shown in Table 2.

P388/AMSA was not crossresistant to any of the alkylating agents and was marginally crossresistant to only one antimetabolite. Crossresistance was observed for all of the drugs tested that are involved in multidrug resistance.

The crossresistance data for P388/DIOHA have been limited mainly to agents in- volved in multidrug resistance. The sensitivity of P388/DIOHA to seven different clini- cal agents is shown in Table 2. The P388/DIOHA line exhibited mixed multidrug resistance—crossresistance to AMSA and VCR but no crossresistance to actinomycin D, ADR, VP-16, or PTX.

The crossresistance profile of P388/VP-16 to 13 different clinical agents is shown in Table 2. The P388/VP-16 line was not crossresistant to any of the alkylating agents or antimetabolites; however, it was crossresistant to all of the drugs tested that are reported to be involved in multidrug resistance.

The sensitivity of P388/CPT to seven different clinical agents is shown in Table 2 (9).

P388/CPT was not crossresistant to any of these agents.

The effect of 21 different clinical agents on P388/VCR is shown in Table 2. The P388/

VCR line was crossresistant to three of the agents—MMC, DDPt (marginal), and vin- blastine. Unexpectedly, P388/VCR was not crossresistant to many of the drugs tested that are involved in multidrug resistance (e.g., actinomycin D, ADR, VP-16, AMSA, mitoxan- trone, and PTX).

The crossresistance data for P388/PTX have been limited to agents involved in multidrug resistance. The sensitivity of P388/PTX to three different clinical agents is shown in Table 2. The P388/PTX line was crossresistant to drugs that are involved in multidrug resistance (ADR, VP-16, and VCR).

4. CONCLUSIONS

Southern Research Institute has evaluated over 100 clinically useful antitumor drugs or new candidate antitumor agents in vivo against 24 drug-resistant P388 leukemias. We have presented here a portion of the results from those studies. Analysis of these data has revealed (1) possible noncrossresistant drug combinations and (2) possible mechanisms of drug resistance.

Schabel and coworkers (5) observed that except for crossresistance to other drugs with

a similar chemical structure and/or biological function, resistance to one drug usually did

not result in resistance to other drugs, particularly those of other functional classes. The

additional data presented here confirm that original observation. P388/CPA, P388/DDPt,

and P388/BCNU were not crossresistant to the alkylating agents tested (except for the

marginal crossresistance of P388/CPA to MMC); P388/CPA and P388/DDPt were not

crossresistant to antimetabolites, DNA-binding agents, or tubulin-binding agents. In

contrast, P388/L-PAM was crossresistant to several of the alkylating agents and to

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representatives of the other functional classes; P388/MMC was crossresistant to most of the DNA- and tubulin-binding agents. The spectrum of crossresistance of an alkylating agent will depend on the individual agent.

P388/MTX was not crossresistant to the other clinical agents tested, whereas P388/5- FU was crossresistant to MTX. Similar to that observed for P388/L-PAM, P388/ARA- C was crossresistant to other antimetabolites and to representatives of two other functional classes. The spectrum of crossresistance of an antimetabolite will also depend on the individual agent.

Schabel and coworkers (5) also noted that crossresistance profiles were variable for the leukemia lines selected for resistance to large polycyclic anticancer drugs. P388/ACT- D, P388/ADR, P388/AMSA, P388/DIOHA, P388/VP-16, P388/CPT, and P388/VCR were not generally crossresistant to alkylating agents or antimetabolites. However, the crossresistance profiles to DNA- and tubulin-binding agents were quite variable. P388/

ADR was crossresistant to all the listed agents, whereas P388/VCR was crossresistant only to vinblastine, and P388/CPT was not crossresistant to any of the tested agents.

Generally, P388/ACT-D, P388/AMSA, P388/VP-16, and P388/PTX were crossresistant to DNA- and tubulin-binding agents, whereas P388/DIOHA exhibited a mixed multidrug resistance.

The crossresistance profile for P388/ADR was almost identical to that for another P388/ADR line selected for resistance in vivo (53). The crossresistance profiles for P388/

AMSA and P388/DIOHA were similar to those reported earlier by Johnson and cowork- ers for the same lines (7,8). Notable differences with respect to agents involved in multidrug resistance were the appearance of crossresistance for our P388/AMSA to actinomycin D and VCR and the appearance and disappearance of crossresistance for our P388/DIOHA to VCR and ADR, respectively.

Six of the 16 drug-resistant leukemias exhibited collateral sensitivity to one or more drugs. These observations of collateral sensitivity suggest that a combination of one of the six drugs plus one of the corresponding agents for which collateral sensitivity was observed might exhibit therapeutic synergism. We have evaluated six such combinations and have observed therapeutic synergism for five of the combinations: ara-C + 5-FU, DDPt + fludarabine, DDPt + AMSA (unpublished results), CPA + DDPt (54), and DDPt + mitoxantrone (54). As always, none of the above approaches may be applied clinically without caution and concern for the recognized gap between preclinical prediction and clinical validation.

Examination of the crossresistance profiles has provided some insights into the mecha- nisms of resistance that are operative in some of these 16 drug-resistant P388 leukemias.

The crossresistance of P388/CPA to alkylating agents of different chemical classes sug- gests that a mechanism of resistance other than aldehyde dehydrogenase is operative.

Furthermore, the lack of crossresistance of P388/CPA to DDPt or L-PAM argues against the involvement of GSH or other thiols in the resistance. The crossresistance of P388/L- PAM to agents of different chemical classes and with different mechanisms of action suggests that another mechanism of resistance besides elevated GSH levels is operative.

Whereas elevated GSH levels could explain the crossresistance to alkylating agents, it is

not clear how elevated GSH levels would account for resistance to agents of the other

functional classes. The observation of collateral sensitivity for P388/DDPt to two agents

that have been reported to interact with DNA topoisomerase II suggests the possible

involvement of the latter in DDPt resistance. Studies in our laboratories have shown that

P388/DDPt cells have more DNA topoisomerase II activity and protein than parental P388/0 cells (unpublished results). Increased DNA topoisomerase II activity has been reported for a DDPt-resistant human small cell lung carcinoma cell line (55). The re- ported elevations in GSH are not apparently sufficient to result in crossresistance of P388/

DDPt to L-PAM.

The reported mechanisms of resistance to ara-C have involved the uptake and metabo- lism of ara-C. However, the crossresistance of P388/ARA-C to several alkylating agents and one tubulin-binding agent suggests that another mechanism of resistance is involved.

The variability of the crossresistance profiles for P388/ACT-D, P388/ADR, P388/

AMSA, P388/DIOHA, P388/VP-16, and P388/VCR to large polycyclic anticancer drugs prompted us to characterize biochemically these drug-resistant leukemias with respect to amplification of the mdr1 gene, level of the 4.5-kb transcript, and level of P-glycoprotein.

Preliminary studies have shown that our P388/ADR, which exhibits multidrug resis- tance, has increased expression of the mdr1 gene, confirming the results reported for another in vivo selected P388/ADR leukemia (33). However, P388/VP-16, which exhib- its comparable multidrug resistance, did not overexpress the mdr1 gene. P388/ACT-D and P388/AMSA, which exhibit multidrug resistance (although not as complete as P388/

ADR), did not overexpress the mdr1 gene. P388/DIOHA, which exhibits a mixed multidrug resistance, also did not overexpress the mdr1 gene. Finally, P388/VCR, which does not exhibit multidrug resistance, did not overexpress the mdr1 gene. Therefore, in vivo multidrug resistance does not require increased expression of the mdr1 gene. Similar conclusions have been made for in vitro multidrug resistance, which has been called

“atypical” multidrug resistance (56).

In conclusion, the in vivo crossresistance profiles of 16 drug-resistant P388 leukemias to 22 clinically useful antitumor drugs have enabled the identification of possible noncrossresistant drug combinations and of insights into the mechanisms of resistance operative in some of the drug-resistant leukemias.

ACKNOWLEDGMENTS

The majority of this work was supported by contracts with the Developmental Thera- peutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Insti- tute. The studies of gemcitabine and P388/VP-P388/VP-16 leukemia were supported by Eli Lilly and Company and by Burroughs Wellcome Company, respectively. The author gratefully acknowledges the technical assistance of the staff of the Cancer Therapeutics and Immunology Department. J. Tubbs assisted with data management, and K. Cornelius prepared the manuscript.

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