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Cognitive impairment is a frequent adverse effect of cancer and its therapies. As neuropsychological assessment is not often standard of care for patients with non-CNS disease, efficient, practical assessment tools are required to track cognition across the disease course. We examined cognitive functioning using a web-based cognitive testing battery to determine if it could detect differences between patients with cancer and controls.We enrolled 22 patients with multiple myeloma (MM) or non-Hodgkin lymphoma (NHL) and 40 healthy controls (mean age = 56 ± 11 years, 52% male). Participants completed the BrainCheck cognitive testing battery and online versions of select measures from the Patient Reported Outcome Measures Information System (PROMIS) during a video conference. MANOVA was used to compare BrainCheck and PROMIS scores between groups controlling for age and sex. An exploratory linear regression analysis was conducted within the cancer group to determine potential contributors to cognitive functioning.All participants except for one control completed the online assessment measures without difficulty. Compared to controls, the cancer group demonstrated significantly lower scores in objective and subjective cognitive function, physical functioning, and social role performance and elevated fatigue scores. Corticosteroid treatment, immunotherapy, lower physical functioning, lower income, and older age significantly contributed to lower cognitive function (adjusted RRemote assessment of cognitive and psychosocial functioning is feasible with patients with cancer following treatments. The BrainCheck cognitive testing battery has the potential to detect differences in cognition between patients with cancer and controls.

Tumor relapse and drug resistance are major factors that limit the curability of multiple myeloma (MM). New regimens have improved overall MM survival rates, but patients with high-risk features continue to have inferior outcomes. Chromosome 17p13 deletion (del17p) which includes the loss of the TP53 gene is a high-risk cytogenetic abnormality and is associated with poor clinical outcomes due to relatively short remissions and the development of pan-drug resistant disease. Increased relapse rates suggest that del17p enhances clonogenic growth, and we found that the loss of p53 increased both the frequency and drug resistance of tumor-initiating MM cells (TICs). Subsequent RNA sequencing (RNA-seq) studies demonstrated significant activation of the Notch signaling pathway and upregulation of inhibitor of DNA binding (ID1/ID2) genes in p53-knock out (p53-KO) cells. We found that the loss of ID1 or HES-1 expression or treatment with a gamma-secretase inhibitor (GSI) significantly decreased the clonogenic growth of p53-KO, but not p53 wild-type cells. GSI treatment in a small set of MM specimens also reduced the clonogenic growth in del17p samples but not in non-del17p samples. This effect was specific as overexpression of the Notch intracellular domain (NICD) rescued the effects of GSI treatment. Our study demonstrates that the Notch signaling and ID1 expression are required for TIC expansion in p53-KO MM cells. These findings also suggest that GSI may be specifically active in patients with p53 mutant MM.

The Hedgehog (Hh) signaling pathway is aberrantly activated in a wide variety of human cancers, and recent clinical studies have demonstrated that pathway inhibitors are effective in advanced basal cell carcinoma (BCC). The majority of these agents have been designed to target SMOOTHENED (SMO), a transmembrane regulator of Hh signaling, but subsequent mutations in SMO have been found to generate drug resistance. In other cancers, oncogenic events that bypass SMO may activate canonical Hh signaling, and SMO antagonists have not demonstrated significant activity in several diseases. Therefore, alternative strategies targeting the Hh pathway downstream of SMO may have clinical utility. Liver X receptors (LXR) regulate cholesterol and fatty acid homeostasis, and LXR activation can inhibit the Hh pathway in normal mouse embryonic fibroblasts. We examined the effects of LXR activation on Hh signaling in human multiple myeloma cells and found that LXR agonists inhibited Hh pathway activity and clonogenic tumor growth in vitro. LXR activation also inhibited putative multiple myeloma cancer stem cells in vivo leading to the loss of tumor initiating and self-renewal potential. Finally, Hh signaling was inhibited downstream of SMO, suggesting that LXR agonists may represent a novel strategy to target pathogenic Hh signaling as well as treat multiple myeloma. Mol Cancer Ther; 13(7); 1873–81. ©2014 AACR.

Supplemental Figure 1. IQGAP1 levels correlate with disease progression and a poor prognosis;Supplemental Figure 2. IQGAP1 levels correlate with RAS pathway activation gene sets;Supplemental Figure 3. The impact of IQGAP1 WW peptide on cell cycle is reversible.

PDF file, 76K, FACS plots for ALDH and CD44 and CD24 controls.

XLSX file, 138K, Gene expression analysis for predictive signatures of response to MRK-003 plus GEM.

S1. Ezrin expression in PDAC CSCs. S2. Effects of Ezrin on self-renewal and migration in PDAC cells. S3. Actin dynamics following Ezrin knock-down and in low-passage PDAC xenografts. S4. Actin dynamics in L3.6pl cells. S5. Treatment of PDAC cells with NSC305787. S6. Effects of Ezrin loss on in vivo tumor initiation.

PDF file, 209K, Representative H&E stained sections from Panc374 xenograft.

XLSX file, 105K, Gene expression analysis for predictive signatures of response to MRK-003.

PDF file, 186K, Baseline gene expression profiles identify signatures of response to single agent MRK-003 and the combination of MRK-003 and GEM.

PDF - 177K, Figure S1. LXR expression in myeloma cell lines. Figure S2. Induction of LXR target genes by non-steroidal LXR agonists. Figure S3. Effects of LXR agonists on clonogenic myeloma growth. Figure S4. Impact of T0901317 on myeloma cell growth and survival. Figure S5. Dose-response relationships of T0901317 in U266 cells. Figure S6. Gene knock-down by siRNA. Figure S7. Effects of LXR agonists on myeloma tumor initiating cells. Figure S8. In vivo activation of LXR target genes.

Supplementary Data from Ligand-dependent Notch Signaling Is Involved in Tumor Initiation and Tumor Maintenance in Pancreatic Cancer

Purpose: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated.Experimental Design: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the γ-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance.Results: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase–expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase–expressing cells in pancreatic cancer initiation.Conclusions: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.

Supplementary Figure 1 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1, where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study, RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies, flow cytometry, and chromatin immunoprecipitation identified two candidate stem cell marker genes, CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1), to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors, we detected a relatively abundant CD133high-ASCL1high-ALDH1high subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133high subpopulation depended on continued ASCL1 expression. Whereas CD133high cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor, CD133low cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity, including variation in key regulatory factors such as ASCL1, can modulate tumorigenicity in SCLC. [Cancer Res 2009;69(3):845–54]

Supplementary Table 1 and Figure Legend from Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers

Supplementary Figure 1 from Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers

PDF file - 1480K, Figure S1. Double knock in clones have single mutant PIK3CA alleles and equivalent expression of mutant and wild type PIK3CA and form aberrant structures in 3D culture. Figure S2. DKI xenografts retain the same number of KRAS and PIK3CA alleles. Figure S3. Knock out of mutant PIK3CA affects tumorigenicity in nude mouse xenograft assays. Figure S4. DKI cell lines show phosphorylation of Akt and Erk in physiologic but not supra-physiologic concentrations of EGF. Figure S5. DKI cells have increased phosphorylation of S6 ribosomal protein but not 4E-BP1. Figure S6. DKI cells are sensitive to the MEK inhibitor U0126 but not to rapamycin and show differential effects on phosphorylation of Akt, Erk, p70S6K and p90RSK. Figure S7. Transgene expression of mutant PIK3CA cDNA with and without RBD mutations in MCF- 10A and KRAS G12V knock in cell lines. Figure S8. Pdk1 activity is increased in DKI cells.

Supplementary Table 3 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Supplementary Figure 3 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

Supplementary Table 1 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

Supplementary Figures 1-6, Table 1B, 2, 4-6 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Supplementary Figure 2 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of “primary” tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced “ALDH-high” cells by ∼3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce “bulk” tumor size. [Cancer Res 2007;67(5):2187–96]

Supplementary Table 1 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Supplementary Figure 4 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

Supplementary Figure Legends 1-4 from Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

SLC25A51 imports oxidized NAD+into the mitochondrial matrix and is required for sustaining oxidative metabolism in human mitochondria. We observed that higher expression of SLC25A51 correlated with poorer survival in Acute Myeloid Leukemia (AML) patient data. Given AML’s dependency on oxidative cell metabolism, we sought to determine the role SLC25A51 may serve in this disease. We found that depleting SLC25A51 in AML cells led to increased apoptosis, as well as prolonged survival in a xenograft model. Metabolic flux analyses indicated that depletion of SLC25A51 shunted flux away from oxidative pathways and promoted glutamine utilization for reductive carboxylation to support aspartate production. Consequently, SLC25A51 loss sensitized AML cells to glutamine deprivation and glutaminase inhibitor CB-839. Together, the work highlights connections between SLC25A51 and oxidative mitochondrial flux in AML. We identified a rationale for targeting SLC25A51 in myeloid cancers with potential for a therapeutic window, especially when coupled with glutaminase inhibition.Statement of significanceThis investigation describes an approach to directly modulate the tricarboxylic acid cycle as a potential vulnerability in oxidative tumors. Using AML models, the work is an inaugural look into SLC25A51’s role supporting oxidative mitochondrial metabolism and identifies SLC25A51 levels as a potential marker for stratification of AML.

SLC25A51 imports oxidized NAD+ into the mitochondrial matrix and is required for sustaining oxidative metabolism in human mitochondria. We observed that higher expression of SLC25A51 correlated with poorer survival in Acute Myeloid Leukemia (AML) patient data. Given AML’s dependency on oxidative cell metabolism, we sought to determine the role SLC25A51 may serve in this disease. We found that depleting SLC25A51 in AML cells led to increased apoptosis, as well as prolonged survival in a xenograft model. Metabolic flux analyses indicated that depletion of SLC25A51 shunted flux away from oxidative pathways and promoted glutamine utilization for reductive carboxylation to support aspartate production. Consequently, SLC25A51 loss sensitized AML cells to glutamine deprivation and glutaminase inhibitor CB-839. Together, the work highlights connections between SLC25A51 and oxidative mitochondrial flux in AML. We identified a rationale for targeting SLC25A51 in myeloid cancers with potential for a therapeutic window, especially when coupled with glutaminase inhibition. Citation Format: Mu-Jie Lu, Jonathan Busquets, Valeria Impedovo, Yu-Tai Chang, William Matsui, Stefano Tiziani, Xiaolu Cambronne. SLC25A51 impacts drug sensitivity in AML cells by sustaining mitochondrial oxidative flux [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A42.

Simple Summary The Hedgehog signaling pathway is aberrantly activated in many myeloid malignancies, and pathway inhibition is clinically beneficial in specific patients with acute myeloid leukemia. However, even with the approval of these agents, the role of Hedgehog signaling in other myeloid disorders is less clear. In this review, we summarize the laboratory studies that have examined Hedgehog signaling in normal and malignant hematopoiesis as well as the clinical studies that have been carried out in several myeloid leukemias. Finally, we explore potential strategies to further expand the use of pathway inhibitors as therapies for these diseases. Abstract Myeloid malignancies arise from normal hematopoiesis and include several individual disorders with a wide range of clinical manifestations, treatment options, and clinical outcomes. The Hedgehog (HH) signaling pathway is aberrantly activated in many of these diseases, and glasdegib, a Smoothened (SMO) antagonist and HH pathway inhibitor, has recently been approved for the treatment of acute myeloid leukemia (AML). The efficacy of SMO inhibitors in AML suggests that they may be broadly active, but clinical studies in other myeloid malignancies have been largely inconclusive. We will discuss the biological role of the HH pathway in normal hematopoiesis and myeloid malignancies and review clinical studies targeting HH signaling in these diseases. In addition, we will examine SMO-independent pathway activation and highlight potential strategies that may expand the clinical utility of HH pathway antagonists.

Objective Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. Methods We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. Results Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. Conclusions Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

Post-transplant cyclophosphamide (PTCy) can be used as the sole immunosuppression after myeloablative conditioning (MAC) for HLA-matched bone marrow transplantation (BMT). However, the effects of graft-versus-host disease (GVHD) with this platform are undefined. We retrospectively analyzed 298 consecutive adult patients with hematologic malignancies who engrafted after MAC HLA-matched sibling donor (MSD; n = 187) or HLA-matched unrelated donor (MUD; n = 111) T-cell-replete BMT with PTCy 50 mg/kg on days +3 and +4. After MSD and MUD BMT, 35% and 57% of patients, respectively, developed grade II acute GVHD (aGVHD) by 100 days, 11% and 14% grade III to IV aGVHD by 100 days, and 9% and 16% chronic GVHD (cGVHD) by 1 year. In landmark analyses at 100 days after HLA-matched BMT, 4-year overall survival (OS) and progression-free survival (PFS) were 57% (95% confidence interval [CI], .49 to .67) and 40% (95% CI, .31 to .51) in patients without grades II to IV aGVHD, and 68% (95% CI, .59 to .78) and 54% (95% CI, .44 to .65) in patients with grade II aGVHD. In adjusted time-dependent multivariable analyses, grade II aGVHD was associated with improved OS (hazard ratio, .58; 95% CI, .37 to .89; P = .01) and PFS (hazard ratio, .50; 95% CI, .34 to .74; P.001) after HLA-matched BMT with PTCy. The ability of PTCy to limit grades III to IV aGVHD and cGVHD while maintaining grade II aGVHD may contribute to its effectiveness, and further attempts to reduce aGVHD may be detrimental.

Self-renewal maintains the long-term clonogenic growth that is required for cancer relapse and progression, but the cellular processes regulating this property are not fully understood. In many diseases, self-renewal is enhanced in cancer stem cells (CSC), and in pancreatic ductal adenocarcinoma (PDAC), CSCs are characterized by the surface expression of CD44. In addition to cell adhesion, CD44 impacts cell shape and morphology by modulating the actin cytoskeleton via Ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family of linker proteins. We examined the expression of Ezrin in PDAC cells and found higher levels of both total and activated Ezrin in CSCs compared with bulk tumor cells. We also found that the knockdown of Ezrin in PDAC cells decreased clonogenic growth, self-renewal, cell migration, and CSC frequency in vitro as well as tumor initiation in vivo. These effects were associated with cytoskeletal changes that are similar to those occurring during the differentiation of normal stem cells, and the inhibition of actin remodeling reversed the impact of Ezrin loss. Finally, targeting Ezrin using a small-molecule inhibitor limited the self-renewal of clinically derived low-passage PDAC xenografts. Our findings demonstrate that Ezrin modulates CSCs properties and may represent a novel target for the treatment of PDAC. Implications: Our findings demonstrate that Ezrin modulates CSCs' properties and may represent a novel target for the treatment of PDAC.

Background: Despite the introduction of novel therapies, the majority of the multiple myeloma (MM) patients eventually relapse and develop chemoresistance, and the factors responsible for disease progression are not fully understood. Relapse suggest that MM cells that are resistant to chemotherapy and have the clonogenic growth potential to produce tumor cells exist, and we have found that tumor cells lacking the plasma cell antigen CD138 are enriched in these properties. Understanding the mechanisms regulating these MM cancer stem cells (CSCs) may lead to novel targeting strategies. Aberrant translation is a widespread characteristic of tumor cells and plays a major role in drug resistance. Thus, therapeutic agents that target translation hold promise as novel anti-cancer drugs. MM cells are characterized by heightened protein production in the form of immunoglobulin (Ig) and are highly dependent on proteolytic pathways such as the proteasome to maintain protein homeostasis. As a consequence, proteasome inhibitors (PIs) such as bortezomib (BTZ) have been established as a frontline therapy for the disease. Immature plasma cells with diminished Ig production are resistant to BTZ, and patients with low Ig producing tumors are more likely to relapse. Therefore, low protein synthesis may confer growth advantages to cancer cells such as chemoresistance and enhanced tumor regrowth potential. Methods & Results: To examine alterations in the translational landscape in therapy resistant MM, we treated MM cells with BTZ, and found that eukaryotic translation initiation factor-5 (eIF5) was highly upregulated. Importantly, over-expression of eIF5 led to increased chemotherapy resistance and enhanced clonogenicity. O-propargyl-puromycin incorporation showed a global translational reduction in cells over-expressing eIF5. Matched transcriptional RNA sequencing (RNA-seq) and ribosome profiling revealed that a subset of genes, including MSI2 and EZH1, escaped translation suppression by maintaining efficient translation in eIF5 over-expressing cells. In addition, clonogenic (CD138neg) MM cells exhibited increased eIF5 expression and decreased protein synthesis. Discussion: Understanding the mechanisms of chemoresistance in the cell populations responsible for residual disease and re-growth may lead to novel targeting strategies. Recent studies have indicated that stem cell behavior is coupled to protein synthesis regulation. However, the role of translational control in the maintenance of tumor-initiating cells has been explored only recently. Studies have implicated deregulation of initiation factors in various types of cancer. However, the role of eIF5 in cancer remains elusive. Our data reveals, for the first time, a novel eIF5-directed translational program that enhances chemoresistance and tumor-initiating potential in MM cells, making it a promising therapeutic target. Citation Format: Kiera Rycaj, Eun-Hee Park, Tori Tonn, Qiuju Wang, Can Cenik, Christian Gocke, William Matsui. A novel translation program regulates tumor-initiating potential and chemoresistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3967.

Multiple myeloma (MM) is a plasma cell malignancy and most patients eventually succumb to the disease. Chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) on MM cells have shown high-response rates, but limited durability. CD229/LY9 is a cell surface receptor present on B and T lymphocytes that is universally and strongly expressed on MM plasma cells. Here, we develop CD229 CAR T cells that are highly active in vitro and in vivo against MM plasma cells, memory B cells, and MM-propagating cells. We do not observe fratricide during CD229 CAR T cell production, as CD229 is downregulated in T cells during activation. In addition, while CD229 CAR T cells target normal CD229high T cells, they spare functional CD229neg/low T cells. These findings indicate that CD229 CAR T cells may be an effective treatment for patients with MM., CD229 is expressed on the surface of multiple myeloma cells, as well as B and T lymphocytes. Here, the authors engineer CD229-specific CAR T cells and, using patient samples and mouse models, show that treatment with these cells reduces tumour burden and results in limited targeting of T cells.

Myelodysplastic syndromes (MDSs) are stem cell disorders with risk of transformation to acute myeloid leukemia (AML). Gene expression profiling reveals transcriptional expression of GLI1, of Hedgehog (Hh) signaling, in poor-risk MDS/AML. Using a murine model of MDS we demonstrated that constitutive Hh/Gli1 activation accelerated leukemic transformation and decreased overall survival. Hh/Gli1 activation resulted in clonal expansion of phenotypically defined granulocyte macrophage progenitors (GMPs) and acquisition of self-renewal potential in a non-self-renewing progenitor compartment. Transcriptome analysis of GMPs revealed enrichment in gene signatures of self-renewal pathways, operating via direct Gli1 activation. Using human cell lines we demonstrated that in addition to canonical Hh signaling, GLI1 is activated in a Smoothened-independent manner. GLI1 knockdown or inhibition with GANT61 resulted in decreased proliferation and clonogenic potential. Our data suggest that GLI1 activation is frequent in MDS during disease progression and inhibition of GLI1 is an attractive therapeutic target for a subset of patients.

With post-transplantation cyclophosphamide (PTCy) as graft-versus-host disease (GVHD) prophylaxis, nonmyeloablative HLA-haploidentical (NMA haplo) and HLA-matched blood or marrow transplantation (BMT) have comparable outcomes. Early discontinuation of immunosuppression may reduce the risk of relapse and improve immune reconstitution, but may increase the risk of GVHD. We conducted a prospective trial of NMA haplo BMT for patients with hematologic malignancies (median age, 61 years), evaluating the safety of early discontinuation of tacrolimus. All patients received T cell-replete bone marrow followed by high-dose PTCy, mycophenolate mofetil, and tacrolimus. Tacrolimus was prespecified to stop without taper at day +90, +60, or +120, contingent on having ≥5% donor T cells, no relapse, and no grade II-IV acute or significant chronic GVHD. Safety stopping rules were based on ≥5% graft failure, ≥10% nonrelapse mortality (NRM), or a ≥20% combined incidence of severe acute and chronic GVHD from the tacrolimus stop date through day +180. Of the 47 patients in the day +90 arm, 23 (49%) stopped tacrolimus as planned. Of the 55 patients in the day +60 arm, 38 (69%) stopped as planned. Safety stopping criteria were not met. In both arms, at day +180, the probability of grade II-IV acute GVHD was

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate less than 10%. Current therapies consisting of cytotoxic chemotherapeutic agents are not effective in most patients with metastatic PDAC, and the impact of new strategies, including immunotherapies, has not been established. Previous studies have shown that inhibition of focal adhesion kinase (FAK) modulates the PDAC immunosuppressive tumor microenvironment (TME), and sensitizes tumors to immune checkpoint blockade. However, the role of FAK in regulating tumor cell intrinsic resistance to immunotherapy is not well understood. We and others previously demonstrated FAK regulates PDAC cancer stem cells (CSCs) activity including self-renewal, tumor initiation, and drug resistance. These CSCs are responsible for metastatic dissemination and resistance to multiple therapies, including immunotherapy. In this study, we evaluated the sensitivity of PDAC cells, including CSCs to cytotoxic T cells, and we also analyzed the impact of FAK inhibition on the immunogenicity and the response to cytotoxic T cell activity of PDAC CSCs. Methods: To understand how FAK regulates tumor intrinsic resistance to antigen-specific cytotoxic T cells, we utilized OT-I T cells (OT-I cells) that specifically recognize a peptide fragment of ovalbumin (ova) within the context of MHC-I. Mouse KPC pancreatic cancer cell lines expressing ova were generated and co-cultured with cytotoxic CD8+ OT-I cells in vitro. We analyzed the effect of FAK in regulating bulk cells sensitivity to antigen-specific cytotoxic T cells and clonogenic growth of CSCs by flow cytometry, MTT assay and colony formation assay using CRISPR-Cas9 and small molecule FAK inhibitors. We also analyzed the expression of surface proteins that mediate interactions with T cells (class I MHC) in bulk cells and CSCs after FAK inhibition. Results: Loss of FAK activity decreased CSCs activity (self-renewal, colony formation) in PDAC mouse cell lines, which is consistent with our previous data. Using antigen-specific OT-I co-culture model, we found that CSCs are resistant to cytotoxic T cells anti-tumor activity compared to bulk tumor cells. We also found inhibition of FAK activity not only enhances PDAC cells to cytotoxic T cell activity, but also sensitized CSCs to T cell killing. Studies in both human and mouse PDAC cell lines demonstrated decreased MHC-I expression in CSCs compared to the bulk tumor cells which could be reversed by FAK inhibition. Conclusion: We have demonstrated that CSCs are resistant to antigen-specific cytotoxic T cells, and FAK inhibition in CSCs can sensitize these cells to anti-tumor T cell activity that is associated with changes in the expression of factors involved in antigen presentation. Our study provides novel insights into the unique immunogenicity of CSCs and the role of CSCs in regulating tumor intrinsic immunotherapy resistance in PDAC. Our findings also lead to the identification of FAK as a novel therapeutic target for CSCs to improve immunotherapy efficacy in PDAC. Citation Format: Yezi Zhu, Lyndsey Sandow, William Matsui. Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-004.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature and thick, desmoplastic stroma, PDAC tumor cells are challenged with meeting a high demand for lipids in a hypoxic, lipid-poor environment. Cancer cells respond to lipid conditions through sterol regulatory element-binding proteins (SREBPs). SREBPs are master transcriptional regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. In this study, we assess the requirement of this pathway using both in vitro and in vivo model systems. Furthermore, we target the SREBP pathway using a novel combination therapy of two FDA-approved compounds: dipyridamole and statins. Methods: We acquired four patient-derived pancreatic adenocarcinoma cell lines containing mutations in both Kras and p53 and knocked out SCAP in each cell line followed by gene-based rescue. Functional growth assays were performed in both lipid-poor and lipid-rich media conditions. Subcutaneous and pancreatic orthotopic xenografts were performed in nude mice using these same cell lines. A previously established mouse line, LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1 Cre (KPC) on a C57Bl/6 background, was utilized as a PDAC model. KPC mice lacking Scap in one or both alleles (Sfl/+ or Sfl/fl) were generated. Cohort survival and histologic analysis were performed for all mice, and results plotted on a Kaplan-Meier survival curve. In each patient cell line, the following drugs were applied individually and in combination: Dipyridamole (DP), tetramethyl-DP (TMDP), Fluvastatin, and Simvastatin. Cell viability post-treatment was assessed, and synergy calculated for each combination using SynergyFinder. Results: In lipid-poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wild type or SCAP rescued cells. In tumor xenograft models, SCAP knockout cells exhibited reduced tumor growth and tumor volume when compared to wild-type cells. In the genetically engineered mouse models, KPC mice exhibited a median survival time of 289 days, while KPCSfl/+ mice show a significantly increased median survival time of 425 days. Additionally, KPCSfl/fl mice did not develop invasive PDAC. Cells treated individually with DP, TMDP and both statins exhibit lipid-dependent growth defects in all cell lines tested. In combination, DP with either statin as well as TMDP with either statin demonstrate synergy in lipid-poor conditions. Conclusions: Our results demonstrate that loss of SCAP in PDAC tumor cells alters the growth capability both in vitro and using mouse xenograft models. Additionally, heterozygous loss of Scap in the KPC mouse model significantly increased survival. Finally, dipyridamole works in synergy with statins to alter growth of PDAC tumor cells. These findings suggest that targeting the SREBP pathway has significant therapeutic potential in PDAC. Citation Format: Stephanie Myers, Meredith McGuire, Wei Shao, Chine Liu, Theodore Ewachiw, Zeshaan Rasheed, William Matsui, Toni Sepalla, Richard Burkhart, Peter Espenshade. Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-009.

Large alternative donor pools provide the potential for selecting a different donor for a second allogeneic (allo) bone or marrow transplant (BMT). As HLA disparity may contribute to the graft-versus-tumor effect, utilizing new mismatched haplotype donors may potentially improve the antitumor activity for relapsed hematologic malignancies despite a previous alloBMT. Data from patients who received a second alloBMT for relapsed hematologic malignancies at Johns Hopkins were analyzed. Outcomes were compared between patients who received a second allograft with the same MHC composition and those who received an allograft with a new mismatched haplotype. Loss of heterozygosity analysis was performed for patients with acute myeloid leukemia (AML) whose first allograft was haploidentical. Between 2005 and 2015, 40 patients received a second BMT for a relapsed hematologic malignancy. The median follow-up is 750 (range, 26 to 2950) days. The median overall survival (OS) in the cohort is 928 days (95% confidence interval [CI], 602 to not reached [NR]); median event-free survival (EFS) for the cohort is 500 days (95% CI, 355 to NR). The 4-year OS is 40% (95% CI, 25% to 64%), and the 4-year EFS is 36% (95% CI, 24% to 55%). The cumulative incidence of nonrelapsed mortality by 2 years was 27% (95% CI, 13% to 42%). The cumulative incidence of grade 3 to 4 acute graft-versus-host disease (GVHD) at 100 days was 15% (95% CI, 4% to 26%); the cumulative incidence of extensive chronic GVHD at 2 years was 22% (95% CI, 9% to 36%). The median survival was 552 days (95% CI, 376 to 2950+) in the group who underwent transplantation with a second allograft that did not harbor a new mismatched haplotype, while it was not reached in the group whose allograft contained a new mismatched haplotype (hazard ratio [HR], .36; 95% CI, .14 to .9; P = .02). EFS was also longer in the group who received an allograft containing a new mismatched haplotype, (NR versus 401 days; HR, .50; 95% CI, .22 to 1.14; P = .09). Although the allograft for this patient's second BMT contained a new mismatched haplotype, AML nevertheless relapsed a second time. Second BMTs are feasible and provide a reasonable chance of long-term survival. An allograft with a new mismatched haplotype may improve outcomes after second BMTs for relapsed hematologic malignancies.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1−/− mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK’s effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.