Thesis (M.A.)--Boston University

Thesis (M.D.)--Boston University

The murine myeloid leukaemias employed in this study were induced in male CBA/H mice following irradiation with sublethal doses of X-ray. The responses of these leukaemic cell lines and normal (murine) bone marrow cells to cytosine arabinoside and mitoxantrone treatment in vitro were monitored. Both clonogenic, and nonclonogenic chemotherapeutic assays such as radioactive precursor uptake, dye-exclusion assay and autoradiography were employed to determine drug-induced cell lethality. In addition, the in vitro proliferative responses of the leukaemic cell lines and normal bone marrow cells to growth factors were determined using a [3H]thymidine uptake assay. Both cytarabine and mitoxantrone were as toxic to normal bone marrow cells as to leukaemic cells from most of the cell lines. Mitoxantrone appears to be more potent than cytarabine against leukaemic cells in vitro. However, it was also more toxic to normal bone marrow cells. Generally, combinations of cytarabine and mitoxantrone resulted in an additive cytotoxic effect. Mitoxantrone appears to have a narrow therapeutic margin when administered to leukaemia bearing mice in vivo. The response of the (SA7) myeloid leukaemic cell line to mitoxantrone was distinctly different from those reported for murine lymphoid leukaemias (P388 and L1210). Doubling the mitoxantrone dose within the therapeutic range was not accompanied by an increase in the number of long-term survivors (cures). Bone marrow cells of cured mice or normal(CBA/H) mice administered low doses of mitoxantrone became less sensitive to subsequent treatment with mitoxantrone in vitro . Doses of mitoxantrone that resulted in loss of protective effect by bone marrow cells of normal mice were toxic to leukaemia bearing mice. Primary and low cell dose transplant myeloid leukaemias were less responsive to growth factors as compared to the high cell dose passages. The SA2 leukaemic cell line grew in vitro without requirement for growth factors. However, no growth was observed in serum-free medium which suggests that serum was providing the stimulus for in vitro proliferation. Leukaemic bone marrow cells from the SA7 high cell dose passage cell line, were normally responsive to growth factors in vitro. However, at relapse following in vivo treatment with mitoxantrone, the leukaemic cells became significantly (P=0.04) growth factor insensitive. Bone marrow cells of normal mice retained growth factor sensitivity following in vivo treatment with mitoxantrone. Furthermore, bone marrow cells of mice cured of leukaemia by mitoxantrone treatment in vivo were responsive to growth factors. Recovery of growth factor responsiveness occurred when the recurrent leukaemic cells were passaged in normal mice. However, no recovery of growth factor sensitivity was observed when recurrent leukaemic cells were passaged in mice that received a single dose of mitoxantrone (0.75mg/Kg) two days previously. Even after passage in normal mice, the recurrent leukaemic cells were in some cases, significantly (P=0.012) resistant to mitoxantrone treatment in vitro. The degree of resistance appears to depend on the dose of mitoxantrone employed in the treatment of the leukaemia. However, passaging the recurrent leukaemia in mitoxantrone pretreated mice did not increase the level of resistance developed by the leukaemic cells. These results suggest that these myeloid leukaemic cell lines may be useful models for preclinical evaluation of chemotherapeutic agents.

Approximately half of all adults diagnosed with acute leukemia (AL) relapse after standard chemotherapy, highlighting the need for alternative treatment options. We have previously shown that vaccination with irradiated autologous tumour cells infected with an oncolytic virus (OV) can elicit a durable, tumour specific, T cell- mediated response in a mouse model of AL. In the context of this AL infected cell vaccine (ICV) model, infection of autologous cells ex vivo with an OV is essential for stimulating a lasting immune response. While the murine AL line L1210 can be robustly infected with Maraba MG1, creating a potent infected cell culture, this ICV still has room for improvement as ICV-vaccinated mice with high tumour burden still die from leukemia. Therefore, we sought to utilize a genome wide CRISPR-Cas9 screen to identify genetic factors that mediate OV resistance in this model of AL. L1210 cells stably expressing Cas9 were transduced with the mouse GeCKOv2 library, which contains 130,209 gRNAs against 20,611 genes within the mouse genome. Following selection, cells were treated with Maraba MG1 and genomic DNA from resistant populations was sequenced to identify genes enriched in resistant cells relative to mock treated cells. Our screen identified several genes that mediate susceptibility to OV infection including those involved in viral entry (Ldlr), receptor-mediated endocytosis (Atp6v1g2), intracellular signaling (Cav1), the cytoskeleton (Filip1 and Tmod4), as well as autophagy and exosome production (Atg5). We aim to use the findings from this work to improve therapeutic efficacy in otherwise OV resistant tumour models as well as identify biomarkers, to determine the feasibility of administering an ICV using patient derived tumour cells.

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Long-term survival and probable cure have become norms in acute lymphoblastic leukaemia of childhood. The adverse effects of treatment for leukaemia are diverse and complex. In many cases, treatment effects come to light 1 ong after the end of therapy. These so-ca 11 ed 1 ate effects (which are yet obscure and incompletely understood) have become increasingly important as the number of children surviving leukaemia increases. This thesis describes a comprehensive study of leukaemia survivors attending the Oncology Clinic of the Red Cross War Memorial Children's Hospital. The study sample consisted of all leukaemia survivors in long-term remission, disease free and off treatment up to January 1st, 1984. The study is introduced by a chapter which describes acute lymphoblastic leukaemia and pays particular attention to the effects of the primary disease on organs which may subsequently exhibit late effects of treatment. Treatment of acute lymphoblastic leukaemia is described in some detail and the reasons for current treatment strategies are outlined. Individual modalities of treatment are then discussed with reference to their mechanisms of action and potential for damage to non-neoplastic tissue. The study then examines all systems likely to have been damaged during therapy, in order to achieve a comprehensive impression of the late effects of leukaemia treatment. In each chapter, pertinent literature was reviewed up to January 1987. Growth is a major task of childhood. Many chronic diseases are potential causes of growth failure. A longitudinal retrospective study showed that statured growth in leukaemia survivors was stunted during treatment. Catch-up growth did not occur at the end of treatment, although normal growth velocity was resumed. Adult height was expected to be reduced as a result. In addition to temporary stunting of statured growth, leukaemia survivors showed a progressive increase in weight-for-height during treatment. This trend continued after treatment had ended. These changes in weight and height were peculiar to leukaemia survivors. Control groups of children with solid tumours in long term remission showed less stunting during treatment and had catch-up growth after treatment, except when they had undergone spinal i rradi ati on. Normal endocrine function is a prerequisite for normal growth and development. Although growth hormone responses to insulin-induced hypoglycaemia were frequently and significantly abnormal in survivors of childhood leukaemia, these children grew normally once treatment had stopped. Impaired growth hormone secretion appeared to be a marker of hypothalamic damage caused by leukaemia therapy. Testicular and ovarian function was normal in the absence of irradiation of these organs. Thyroid function was normal in leukaemia survivors although a minority showed evidence of hypothalamic damage in their response to thyrotropin releasing hormone. Normal prolactin levels in children showing other hormonal evidence of hypothalamic damage were thought to indicate the selectivity of damage caused by leukaemia treatment. Adrenal control and function were normal in leukaemia survivors. In the absence of a growth disorder, only thyroid status may need long-term assessment in leukaemia survivors. Intellectual development is a further major task of childhood. A sibling-controlled study of intellectual function indicated an intelligence deficit in children surviving leukaemia and its treatment. This deficit was thought to be the consequence of therapy, since children surviving solid tumours showed no such deficit in comparison with their sibling controls. Survivors of childhood leukaemia also had an increased incidence of visual perceptual difficulty and more school prob 1 ems than survivors of solid tumours, particularly in early primary grades. Intellectual outcome and school performance in leukaemia survivors may be improved by early visual perceptual training. Children surviving acute lymphoblastic leukaemia had significantly more minor motor abnormalities than children surviving solid tumours. Minor motor abnormalities were frequently and significantly associated with abnormalities of the brain visualized by computerized tomography. Neurophysiologic measurement (EEG, VER, BAER) did not contribute to the assessment of neurological outcome and correlated poorly with clinical and CT scan findings. A functional assessment of neurological outcome in leukaemia survivors should include a clinical examination for minor motor dysfunction. Some children manifested other organ-specific damage due to chemotherapy or radiotherapy. These isolated cases are discussed in the form of case reports and literature reviews. Patients have received treatment with cytotoxic drugs in addition to standard leukaemia therapy need to be followed for treatment-specific late effects. The psychological outcome of leukaemia survivors was assessed by means of parent interviews and teacher questionnaires. In terms of a low frequency of behaviour problems reported by these observers, psychosocial adaptation in leukaemia survivors vas surprisingly good. Children surviving solid tumours and healthy school children from the same community (the latter from a literature report) had similar frequencies of behavioural problems. In both leukemic children ana solid tumour control patients, certain patterns of family behaviour ~ere predictive of a poor psychological outcome. It appears that an early family assessment may identify families 'at risk'. If needs to be shewn whether such families would benefit from professional psychological support. In the final chapter a 'functional deficit score' is offered as a measure of overall outcome in terms of late effects of therapy. Patients were rated in five categories (growth, intellectual outcome, neurological status, miscellaneous organ damage and psychosocial adaptation) according to the severity of persistent late effects. Children surviving acute lymphoblastic leukaemia were shown to have been more seriously damaged by their treatment than children surviving solid tumours. The difference in overall damage was the consequence of central nervous system injury. Available evidence indicates that this central nervous system injury is caused by radiotherapy (with or ·thought a synergistic effect with i intrathecal 1 methotrexate) given as central nervous system 'prophylaxis'. With few exceptions, leukaemia survivors in this study had received L400 rads of deep x-ray therapy as cranial irradiation. This dosage has since been reduced world-wide. Current cranial irradiation 'prophylaxis' consists of 1800 rad of megavoltage radiotherapy. Fa 11 ow-up studies of survivor cohorts given such radiotherapy should include the measures embodied in the 'functional deficit score' described above.