Tretinoin Enhances the Effects of Chemotherapy in Juvenile Myelomonocytic Leukemia Using an Ex Vivo Drug Sensitivity Assay.

PURPOSE: Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric malignancy with myelodysplastic and myeloproliferative features. Curative treatment is restricted to hematopoietic stem-cell transplantation. Fludarabine combined with cytarabine (FLA) and 5-azacitidine (AZA) monotherapy are commonly used pre-transplant therapies. Here, we present a drug screening strategy using a flow cytometry-based precision medicine platform to identify potential additional therapeutic vulnerabilities. METHODS: We screened 120 dual- and 10 triple-drug combinations (DCs) on peripheral blood (n = 21) or bone marrow (n = 6) samples from 27 children with JMML to identify DCs more effectively reducing leukemic cells than the DCs' components on their own. If fewer leukemic cells survived a DC ex vivo treatment compared with that DC's most effective component alone, the drug effect was referred to as cooperative. The difference between the two resistant fractions is the effect size. RESULTS: We identified 26 dual- and one triple-DC more effective than their components. The differentiation agent tretinoin (TRET; all-trans retinoic acid) reduced the resistant fraction of FLA in 19/21 (90%) samples (decrease from 15% [2%-61%] to 11% [2%-50%] with a mean effect size of 3.8% [0.5%-11%]), and of AZA in 19/25 (76%) samples (decrease from 69% [34%-100+%] to 47% [17%-83%] with a mean effect size of 16% [0.3%-40%]). Among the resistant fractions, the mean proportion of CD38+ cells increased from 7% (0.03%-25%; FLA) to 17% (0.3%-38%; FLA + TRET) or from 10% (0.2%-31%; AZA) to 51% (0.8%-88%; AZA + TRET). CONCLUSION: TRET enhanced the effects of FLA and AZA in ex vivo assays with primary JMML samples.

Successful treatment and integrated genomic analysis of an infant with FIP1L1-RARA fusion-associated myeloid neoplasm.

FIP1L1-RARA-a ssociated neoplasm is a very rare and aggressive disease, with only 3 previously reported cases in the literature. Here, we describe a 9-month-old boy who presented with a FIP1L1-RARA fusion-associated myelodysplastic/myeloproliferative neoplasm-like overlap syndrome, with similarities and distinct features to both acute promyelocytic leukemia and juvenile myelomonocytic leukemia. Using a combined approach of chemotherapy, differentiating agents, and allogeneic hematopoietic stem cell transplant (allo-HCT), this patient remains in remission 20 months after allo-HCT. To our knowledge, this is only the second published pediatric case involving this condition and the only case with a favorable long-term outcome. Given the aggressive disease described in the previously published case report, as well as the successful treatment course described, the combinatorial use of chemotherapy, differentiation therapy, and allo-HCT for treatment of FIP1L1-RARA fusion-associated myeloid neoplasms should be considered.

Rova-T enhances the anti-tumor activity of anti-PD1 in a murine model of small cell lung cancer with endogenous Dll3 expression.

Rovalpituzumab tesirine (Rova-T) offers a targeted therapy for ~85% of SCLC patients whose tumors express DLL3, but clinical dosing is limited due to off-target toxicities. We hypothesized that a sub-efficacious dose of Rova-T combined with anti-PD1, which alone shows a clinical benefit to ~15% of SCLC patients, might elicit a novel mechanism of action and extend clinical utility. Using a pre-clinical murine SCLC tumor model that expresses Dll3 and has an intact murine immune system, we found that sub-efficacious doses of Rova-T with anti-PD1 resulted in enhanced anti-tumor activity, compared to either monotherapy. Multiplex immunohistochemistry (IHC) showed CD4 and CD8 T-cells primarily in normal tissue immediately adjacent to the tumor. Combination treatment, but not anti-PD1 alone, increased Ki67+/CD8 T-cells and Granzyme B+/CD8 in tumors by flow cytometry and IHC. Antibody depletion of T-cell populations showed CD8+ T-cells are required for in vivo anti-tumor efficacy. Whole transcriptome analysis as well as flow cytometry and IHC showed that Rova-T activates dendritic cells and increases Ccl5, Il-12, and Icam more than anti-PD1 alone. Increased tumor expression of PDL1 and MHC1 following Rova-T treatment also supports combination with anti-PD1. Mice previously treated with Rova-T + anti-PD1 withstood tumor re-challenge, demonstrating sustained anti-tumor immunity. Collectively our pre-clinical data support clinical combination of sub-efficacious Rova-T with anti-PD1 to extend the benefit of immune checkpoint inhibitors to more SCLC patients.

Identification and Targeting of Long-Term Tumor-Propagating Cells in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all clinical features of SCLC are linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors and a means to target them in this most fatal form of lung cancer.

A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo.

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.

Initiation and characterization of small cell lung cancer patient-derived xenografts from ultrasound-guided transbronchial needle aspirates.

Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980's, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.

PURPOSE: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. EXPERIMENTAL DESIGN: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. RESULTS: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. CONCLUSIONS: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.

Patient-derived xenografts, the cancer stem cell paradigm, and cancer pathobiology in the 21st century.

Cancer is a heterogeneous disease manifest in many forms. Tumor histopathology can differ significantly among patients and cellular heterogeneity within tumors is common. A primary goal of cancer biologists is to better understand tumorigenesis and cancer progression; however, the complex nature of tumors has posed a substantial challenge to unlocking cancer's secrets. The cancer stem cell (CSC) paradigm for the pathobiology of solid tumors appropriately acknowledges phenotypic and functional tumor cell heterogeneity observed in solid tumors and accounts for the disconnect between drug approval based on response and the general inability of approved therapies to meaningfully impact survival due to their failure to eradicate these most important of cellular targets. First proposed to exist decades ago, CSC have only recently begun to be precisely identified due to technical advancements that facilitate identification, isolation, and interrogation of distinct tumor cell subpopulations with differing ability to form and perpetuate tumors. Precise identification of CSC populations and the complete hierarchy of cells within solid tumors will facilitate more accurate characterization of patient subtypes and ultimately contribute to more personalized and effective therapies. Rapid advancement in the understanding of tumor biology as it exists in patients requires cooperation among institutions, surgeons, pathologists, cancer biologists and patients alike, primarily because this translational research is best done with patient-derived tissue grown in the xenograft setting as patient-derived xenografts. This review calls for a broader change in the approaches taken to study cancer pathobiology, highlights what implications the CSC paradigm has for pathologists and cancer biologists alike, and calls for greater collaboration between institutions, physicians and scientists in order to more rapidly advance our collective understanding of cancer.

Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates.

Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G(0) fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation.